Developmental Dopamine Loss Rewires Striatal Circuits to Promote Locomotion

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Sullivan, Victor M. Martinez Smith, Lupeng Wang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7401124/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2026 Read the published version in Molecular Neurodegeneration → Version 1 posted 11 You are reading this latest preprint version Abstract Background: Motor symptoms of Parkinson’s disease (PD) primarily result from the degeneration of nigrostriatal dopaminergic neurons (DANs), particularly the Aldehyde Dehydrogenase 1A1-positive (ALDH1A1⁺) subpopulation. Pitx3 -deficient mice exhibit selective developmental loss of ALDH1A1⁺ DANs but paradoxically display hyperlocomotion, suggesting compensatory changes in striatal circuitry. The dorsal striatum contains four main types of spiny projection neurons (SPNs): patch (or striosome) and matrix subtypes of both direct-pathway (dSPNs) and indirect-pathway (iSPNs). Activation of patch dSPNs suppresses locomotion by inhibiting ALDH1A1⁺ DANs. Methods: We combined RNAscope in situ hybridization with SPN subtype-specific reporter mice to quantify patch and total dSPNs and iSPNs in Pitx3 -deficient and control mice. Three patch SPN reporter lines ( Kremen1 2A-Cre , Nr4a1 - GFP , and Pdyn IRES-Cre ) were used to map projections. Optogenetic stimulation was performed in freely moving mice to assess the behavioral effects of activating patch dSPNs and iSPNs. Results: Pitx3- deficient mice showed no change in the overall dSPN:iSPN ratio but exhibited a marked shift in the patch dSPN:patchy iSPN ratio, which decreased from 1.7 in control mice to 0.7 in the Pitx3- deficient group. Accordingly, patch dSPN projections to the substantia nigra pars reticulata (SNr) were reduced, whereas patch iSPN projections to the globus pallidus externus (GPe) were enhanced. Notably, while optogenetic stimulation of patch dSPNs and iSPNs suppressed locomotion in control mice, the same stimulation promoted locomotion in Pitx3 -deficient mice. Conclusions: Our findings reveal a selective reorganization of patch SPNs in response to developmental loss of ALDH1A1⁺ DANs, characterized by reduced patch dSPN and enhanced patch iSPN influence. This shift may underlie the paradoxical hyperlocomotion observed in Pitx3 -deficient mice and provides insight into circuit-level adaptations with potential therapeutic relevance for PD. ALDH1A1 Kremen1 dopaminergic neurons Parkinson’s disease Pitx3 striatal projection neurons direct-pathway indirect-pathway optogenetics and neuromodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Patients with Parkinson’s disease (PD) experience progressive motor symptoms, including resting tremor, slowed movement, and impaired posture and balance [ 1 ]. In addition to these motor issues, they often suffer from non-motor symptoms such as depression and dementia [ 2 ]. While medications and surgical interventions can improve motor function, long-term medication can lead to severe side effects, including dyskinesia and impulse control disorders [ 3 , 4 ]. Therefore, there is a continuing need for new mechanistic insights and therapeutic strategies to improve treatment options for the growing number of PD patients [ 5 ]. The motor symptoms of PD are primarily associated with the degeneration of midbrain dopaminergic neurons (DANs) in the substantia nigra compacta (SNc) [ 6 , 7 ], particularly the Aldehyde Dehydrogenase 1A1-positive (ALDH1A1 + ) subset in its ventral tier [ 8 – 11 ]. The ALDH1A1 + SNc DANs accounts for 60–70% DANs in human and rodent SNc and mainly project to the dorsal portion of dorsal striatum [ 8 , 10 , 12 , 13 ]. In naturally occurring Pituitary homeobox 3 ( Pitx3 )-deficient aphakia (or Pitx3 ak/ak ) mice, midbrain DAN differentiation remains unaffected at embryonic day 11.5 (E11.5) [ 14 ]. However, a noticeable reduction in tyrosine hydroxylase (TH)-positive cells destined for the SNc emerges by E12.5 [ 14 ], leading to a significant loss of SNc DANs by postnatal day 3 [ 15 ]. In contrast, DANs in the ventral tegmental area (VTA) remain largely unaffected [ 14 – 17 ]. Notably, the depleted SNc DANs in these mice predominantly belong to the ALDH1A1 + DAN subpopulation [ 18 ]. Interestingly, while acute genetic ablation of ALDH1A1 + DANs in adult mice slows locomotion [ 12 ], the developmental loss of these neurons in Pitx3 ak/ak mice does not decrease nighttime movement [ 19 ]. Instead, it increases daytime activity [ 19 ]. This adaptation likely involves striatal projection neurons (SPNs) in the dorsal striatum, which are the primary targets of midbrain DAN input [ 20 , 21 ]. Investigating these compensatory mechanisms could provide valuable insights into neuromodulatory strategies for addressing PD-related motor deficits. In the dorsal striatum, SPNs are broadly classified into two major subtypes based on their molecular identity and projection targets: direct-pathway SPNs (dSPNs), which express dopamine receptor D1 (DRD1) and project to the internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr), and indirect-pathway SPNs (iSPNs), which express dopamine receptor D2 (DRD2) and project to the external globus pallidus (GPe) [ 22 – 25 ]. Both dSPNs and iSPNs are distributed across two complementary compartments—the patch (or striosome) and the matrix, which differ in anatomical organization, gene expression profiles, and connectivity patterns [ 26 – 30 ]. Notably, SPNs with molecular characteristics of patch neurons can also be found scattered within the matrix compartment; these are referred to as "exo-patch" SPNs [ 31 ]. Since molecularly labeling does not distinguish between canonical patch and exo-patch locations, we use the term “patchy SPNs” to collectively refer to both populations in this study. Among midbrain DANs, ALDH1A1 + DANs in the SNc receive the strongest monosynaptic inhibitory input from patchy dSPNs [ 12 , 32 ]. Recent studies have shown that activation of patchy dSPNs suppresses locomotion via inhibition of ALDH1A1 + DANs and reduction of dopamine release [ 33 – 35 ], whereas patchy iSPNs appear to facilitate movement, highlighting compartment- and cell type-specific functional roles [ 33 , 35 ]. However, it remains unclear how the developmental loss of ALDH1A1 + DANs in Pitx3 ak/ak mice affects the organization and function of SPNs, particularly patchy SPNs. In this study, we examine the structural and functional reorganization of SPNs, with a focus on patchy SPNs, in Pitx3 ak/ak mice. We analyze the composition and distribution of molecularly defined patchy dSPNs and iSPNs in the dorsal striatum and assess their projection patterns to the GPe and SNr. Additionally, we use optogenetic approaches to selectively activate patchy dSPNs and iSPNs to determine their impact on locomotion. Methods Animals All mouse procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Intramural Research Program at the National Institute on Aging (NIA), NIH, and conducted in accordance with institutional and NIH guidelines. All mouse lines were maintained as heterozygotes on a C57BL/6J background. The following strains were obtained from The Jackson Laboratory: Pitx3 ak/ak (Stock No: 000942) [ 17 ], Drd1– tdTomato (Stock No: 016204) [ 36 ], Pdyn IRES−Cre (Stock No: 027958) [ 37 ], and Ai14 (Stock No: 007908) [ 38 ]. The Pitx3 ak/ak mice were backcrossed with C57BL/6J for more than five generations. The Kremen1 2A − Cre knock-in mice [ 33 ] were generated by Shanghai Model Organisms Inc. (Shanghai, China), while the Nr4a1– eGFP (Stock No: 036737-UCD) transgenic mice [ 39 ] were obtained from the Mutant Mouse Resource & Research Centers (MMRRC). To examine the projections of patchy dSPNs and iSPNs, three patchy SPN mouse lines ( Kremen1 2A − Cre , Pdyn IRES−Cre , and Nr4a1– eGFP) were bred into the Pitx3 ak/ak background, and their littermates Pitx3 +/ak were used as control in the experiments. Both male and female mice were used in all experiments. Mice were group-housed (2–5 per cage) under a 12-hour light/dark cycle with ad libitum access to water and standard chow. Behavioral experiments were conducted during the light phase. Littermates were randomly assigned to experimental groups before study onset. RNA In Situ Hybridization and Image Analysis RNA in situ hybridization (RNAscope, ACDBio) was performed to detect Drd1 , Drd2 , and Kremen1 mRNAs in the dorsal striatum of adult C57BL/6J mice. Mice were euthanized with CO₂ inhalation. Brains were fresh-frozen on dry ice and stored at − 80°C. Coronal sections (12 µm) were cut on a cryostat (Leica Biosystems) and stored at − 80°C. RNAscope was performed using the Multiplex Fluorescent Reagent Kit v2 per the manufacturer's instruction. Probes used included: Drd1 (Cat. No. #401901), Drd2 (Cat. No. #406501), and Kremen1 (Cat. No. #425771). Images were acquired on a Zeiss LSM 780 laser scanning confocal microscope with 20× or 40× objectives. Five sections were analyzed spanning a rostro-caudal range from approximately 1.34 mm to − 0.34 mm relative to Bregma. Image analysis was conducted using Imaris v10.0 (Bitplane, Belfast, UK). Dorsal and ventral striatal regions were delineated using the Allen Brain Atlas. Spatial patches were defined as clusters of at least five Kremen1 + SPNs with a minimum density of 200 cells/mm². Channels ( Drd1 , Drd2 , Kremen1 , and DAPI) were analyzed with consistent parameters within each batch; minor adjustments across batches ensured optimal quantification. DAPI + cells were classified as Drd1 + (dSPNs) or Drd2 + (iSPNs) based on mean fluorescence intensity. Overlap with Kremen1 signal was used to quantify patchy dSPNs and iSPNs. Data points represent averages across multiple bilateral striatal sections. Immunohistochemistry Mice were anesthetized with pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed overnight at 4°C, then cryoprotected in 30% sucrose (PBS-buffered) for ≥ 48 hours. Coronal sections (40 µm) were cut and stored in PBS with 4% sodium azide at 4°C. Sections were blocked for 1 hour at room temperature in 10% normal donkey serum, 0.5% BSA, and 0.3% Triton-X-100. They were incubated with primary antibodies overnight or for 48 hours at 4°C, washed (3 × 10 min, PBS), and then incubated with secondary antibodies for 1 hour. Some sections were counterstained with DAPI (0.5 mg/mL, 1 min, Invitrogen, D1306) and mounted using ProLong Gold Antifade Mountant (Life Technologies). Images were captured using a Zeiss LSM 780 or LSM 980 confocal microscope. Primary antibodies used: polyclonal rabbit anti-PITX3 (1:500) [ 40 ], rabbit anti-TH (Pel-Freez Biologicals, P40101; 1:1000), goat anti-ALDH1A1 (R&D system; 1:1000), guinea pig anti-PV (Swant, GP72; dilution 1:1000), goat anti-ChAT (AB144P, Millipore; dilution 1:500) and rat anti-CTIP2 (abcam, ab18465, 1:500). Secondary antibodies (Life Technologies) were selected for appropriate fluorophore spectra. Quantification of TH and ALDH1A1 Neurons Midbrain sections (40 µm) spanning Bregma − 2.70 mm to − 3.80 mm (seven sections per mouse) were stained for TH, ALDH1A1 and DAPI. Z-stack confocal images were captured at 20× magnification. Neurons in the SNc and VTA were quantified using the “Detect Cells ” functions in NeuroInfo (MBF Bioscience). Regions were manually delineated using previously established anatomical landmarks. Total neuron numbers were estimated using “Area Under the Curve” analysis in GraphPad Prism 10. Quantification of TH + and ALDH1A1 + Axon Terminal in the Striatum Five to six striatal sections across a rostro-caudal series (approximately 1.42mm to -0.2 mm relative to Bregma) were co-stained for TH and ALDH1A1. Images were acquired with 10 × objective on the Zeiss LSM780. ALDH1A1 + areas in the dorsal striatum were outlined using intensity threshold in ImageJ. For each section, the percentage of ALDH1A1 + area relative to total striatal area was calculated. Quantification of Nr4a1-eGFP cells in the dorsal striatum In Nr4a1 -eGFP; Drd1 -tdTomato mice on either Pitx3 +/ak or Pitx3 ak/ak backgrounds, striatal sections spanning from Bregma 1.34mm to -0.34mm (5 sections per each mouse) were stained for CTIP2 and imaged using a Zeiss LSM 780 laser scanning confocal microscope with a 10× objective. Dorsal striatal regions were delineated according to the Allen Brain Atlas. Cell quantification was performed using Imaris v10.0 (Bitplane, Belfast, UK). CTIP2 + cells were identified as SPNs and further classified as tdT + (dSPNs) and tdT-negative (iSPNs) based on median fluorescence intensity. Overlap with GFP + signal was used to quantify Nr4a1-eGFP + dSPNs and iSPNs. Minor threshold adjustments were applied across batches to ensure optimal quantification. Quantification of fluorescence intensity and cell density Fluorescence intensity and cell density were quantified using ImageJ. For each mouse, 3–8 striatal or midbrain sections at different Bregma levels were analyzed. For SNr and GPe regions, mean RFP or GFP fluorescence intensity was calculated by delineating the regions of interest based on anatomical landmarks in the Allen Brain Atlas. To assess fluorescence distribution, “Plot Profile” function was used to generate intensity plots along the drawn line. Colocalization analysis between GFP + and PV + signals was performed using the “Coloc2” function in ImageJ to calculate Manders' coefficients M1 and M2, indicating the proportion of GFP signal overlapping with PV and vice versa. For cell density analysis, PV + and ChAT + cells were automatically counted in the dorsal striatum by setting the particle diameter. The counting area was measured, and cell density was calculated as the number of positive cells per mm². Stereotaxic Viral Injection and Optic Fiber Implantation Stereotaxic surgeries were performed under aseptic conditions. Mice (2–4 months old) were anesthetized with 1–2% isoflurane and placed in a stereotaxic frame (Kopf Instruments). A total of 700 nL of AAV was bilaterally injected into the dorsal striatum (AP: +0.9 mm; ML: ±2.2 mm; DV: −2.5 mm) at 75 nL/min using a microinjector (Stoelting). Vectors included AAV1-EF1a-double floxed-hChR2(H134R)-EYFP (#20298) and AAV1-Ef1a-DIO-EYFP (#27056) from Addgene (Watertown, MA, USA). The needle was left in place for 5 minutes post-injection before withdrawn. Incisions were closed and mice recovered in home cages. After three weeks, optical fibers (200 µm core, 0.39 NA; Thorlabs) were bilaterally implanted targeting either the dorsal striatum (AP: +1.0 mm; ML: ±1.5 mm; DV: −2.2 mm to − 2.7 mm) or SNr (AP: −3.1 mm; ML: ±1.5 mm; DV: −4.1 mm DV). Fibers were secured with radiopaque adhesive cement (C&B METABOND, Parkell) and incisions were sealed with Vetbond tissue adhesive (3M). Mice recovered for ≥ 1 week before testing. Open-Field Spontaneous Locomotion Freely moving mice were assessed for spontaneous locomotion via video tracking. Prior to testing, mice were habituated for 30 minutes. The arena (50 × 50 cm, opaque gray) was lit diffusely using an enclosed 20W lamp. Mice were recorded for 30 minutes at 30 Hz from above using a digital camera. For analysis, the arena was divided into a center zone (central 25 × 25 cm) and a surrounding zone. Data on velocity, distance, and time traveled were analyzed with EthoVision XT (Noldus). For beam break tests, mice were placed in a 43 × 23 cm arena with infrared sensors. Chambers were cleaned with 50% ethanol between trials. Optogenetics in the Open Field Test Light delivery was controlled using an LED source and commutator (PlexBright, Plexon) connected via a patch cable (200 µm, 0.39 NA). Connections were made with ceramic sleeves (Thorlabs). Light power (465 nm) was calibrated to 3 mW at the fiber tip (Thorlabs PM100D). For ChR2 activation, 5-ms light pulses were delivered at varying frequencies and durations controlled via a TTL signal generator (OPTG-4, Doric Lenses). Mice were habituated for 30 minutes before testing. The arena (50 × 50 cm, transparent walls) was cleaned between sessions. Mice were recorded from both top and side views (Logitech cameras) at 15 Hz. After a 3-minute baseline, optogenetic stimulation was applied bilaterally in 10 sec ON / 1 min OFF cycles. Video and TTL signals were synchronized using Synapse software (TDT). Locomotion metrics were analyzed in EthoVision XT. Statistical Analysis Statistical analyses were performed in GraphPad Prism 10 and custom MATLAB scripts (MathWorks). Data are presented as mean ± SEM. Specific statistical tests are reported in figure legends. Significance was assessed using two-tailed t-tests and one-way ANOVA, with thresholds of p < 0.05 ( * ), p < 0.01 ( ** ), p < 0.001 ( *** ), and p < 0.0001 ( **** ). Results Pitx3 ak/ak mice exhibit hyperlocomotion despite the loss of ALDH1A1⁺ DANs Consistent with previous findings [ 14 , 16 , 17 ], PITX3 expression was completely absent in midbrain DANs of Pitx3 ak/ak mice ( Supplementary Fig. S1 ). Loss of PITX3 resulted in a selective depletion of ALDH1A1 + DANs in the ventral SNc, whereas ALDH1A1– SNc DANs and VTA DANs remained largely intact (Fig. 1 a, 1 b). Accordingly, ALDH1A1 + axon projections were severely reduced in the dorsal striatum of Pitx3 ak/ak mice (Fig. 1 c-e). Unexpectedly, Pitx3 ak/ak mice displayed hyperactivity in the open-field test compared to littermate controls (Fig. 1 f, 1 g). Mutant mice also spent significantly more time and traveled longer distances in the peripheral zone relative to the center (Fig. 1 h, 1 i), indicative of elevated anxiety-like behavior. This paradoxical increase in locomotor activity despite the loss of ALDH1A1 + DANs suggests a possible developmental compensatory mechanism, potentially involving circuit-level reorganization of SPNs. Significant reduction in patchy dSPNs and increase in patchy iSPNs in Pitx3 ak/ak mice To assess changes in SPN subtypes, we performed multiplexed RNAscope in situ hybridization using probes for Drd1 , Drd2 , and Kremen1 , which label dSPNs, iSPNs, and a subset of patchy SPNs, respectively [ 33 , 41 ]. This approach allowed us to map the composition and distribution of SPN subtypes in the dorsal striatum of Pitx3 ak/ak and littermate control mice (Fig. 2 a, 2 b). Although Pitx3 -deficiency led to a significant reduction in the overall surface area of the dorsal striatum (Fig. 2 c), it did not alter the size or number of patch-like structures (Fig. 2 d, 2 e), nor did it affect the total numbers of dSPNs or iSPNs (Fig. 2 f). Notably, the number of Kremen1 + patchy dSPNs was substantially reduced in Pitx3 ak/ak mice, whereas the number of patchy iSPNs showed a slight increase (Fig. 2 g). Quantification of the proportion of Kremen1 + cells within the total dSPN and iSPN populations revealed opposite trends: the fraction of patchy dSPNs was significantly decreased, while that of patchy iSPNs was increased (Fig. 2 h). As a result, the ratios of patchy dSPNs to patchy iSPNs was reversed, shifting from 1.7 in control mice to 0.7 in Pitx3 ak/ak mice (Fig. 2 i). These findings were corroborated in an independent patchy SPN reporter model, Nr4a1 -eGFP; Drd1 -tdTomato; Pitx3 ak/ak mice, in which patchy SPNs were labeled by GFP [ 39 ], dSPNs by tdTomato (tdT) [ 36 ], and iSPNs identified as CTIP2-positive but tdT-negative (CTIP2 + /tdT – ) cells (Fig. 3 a-e). CTIP2 is a general marker for SPNs [ 42 ]. By contrast, we observed no apparent changes in PV + and ChAT + interneurons in the dorsal striatum of Pitx3 ak/ak mice ( Supplementary Fig. S2 ). Together, these findings from two independent models demonstrate that PITX3 loss selectively alters patchy SPN composition, characterized by a decrease in patchy dSPNs and a relative increase in patchy iSPNs. Reduced SNr projections from patchy dSPNs and increased GPe projections from patchy iSPNs in Pitx3 ak/ak mice To assess the output pathways of patchy SPNs, we generated Kremen1 2A − Cre ; Ai14 bigenic mice on Pitx3 +/ak or Pitx3 ak/ak backgrounds, enabling selective labeling of Kremen1 + patchy SPNs with tdT. In Pitx3 ak/ak mice, axonal projections to the GPe were increased, whereas projections to the SNr were markedly reduced compared to controls (Fig. 4 a). Moreover, patchy SPNs in these mice appeared more dispersed and lacked the characteristic clustered organization within the superficial dorsolateral striatum (DLS) (arrow, Fig. 4 b). The increase in projection was localized primarily to the dorsal GPe (Fig. 4 c); while dendron-bouquet structures, formed by the dendrites of DANs and axons of patchy dSPN [ 43 ], were entirely absent in the SNr (Fig. 4 d). Quantitative analyses of serial GPe and SNr sections confirmed these projection changes ( Fig, 4e, 4f ). Similar alterations in both somatic distribution in the DLS and projection patterns in GPe and SNr were observed in Nr4a1 -eGFP; Pitx3 ak/ak mice (Fig. 5 a-i). Given that dSPNs can send axonal collaterals to the GPe [ 44 ], we next examined whether increased dSPN collateralization might account for the enhanced GPe projections. To test this, we analyzed Pdyn IRES−Cre ; Ai14 mice, in which tdT preferentially labels patchy dSPNs [ 45 , 46 ]. In these mice, PITX3 loss did not affect GPe projections but still led to a reduction in SNr projections (Fig. 6 a-e). To further investigate the contribution of dSPN collaterals, we examined Nr4a1 -eGFP; Drd1 -tdT mice and found no difference in tdT signal intensity in the GPe of Pitx3 ak/ak and control mice ( Supplementary Fig. S3 ). Taken together, these complementary datasets suggest that PITX3 deficiency disrupts the balance of patchy SPN output pathways by reducing SNr projections due to a loss of patchy dSPNs and increasing GPe projections through an expansion of patchy iSPNs. Optogenetic stimulation of patchy SPNs reveals reversed motor effects in Pitx3 ak/ak mice Recent studies have shown that patchy dSPNs suppress locomotion via inhibition of ALDH1A1 + DANs [ 33 , 34 ], whereas patchy indirect pathway SPNs (iSPNs) exert a weaker locomotion-promoting effect [ 33 , 47 ]. Under normal conditions, the net outcome of patchy SPN activation is locomotion inhibition [ 33 ]. To examine the functional consequences of patchy SPN reorganization in Pitx3 ak/ak mice, we performed optogenetic stimulation to activate either both patchy dSPNs and iSPNs or dSPNs alone (Fig. 7 a, 7 b). In control mice, stimulation of both patchy SPN subtypes significantly reduced locomotor velocity (Fig. 7 c, 7 d), consistent with the dominant inhibitory role of patchy dSPNs [ 33 ]. In contrast, the same stimulation paradigm in Pitx3 ak/ak mice resulted in a marked increase in locomotor speed (Fig. 7 c, 7 d), indicating a reversal of functional output. Notably, direct stimulation of patchy dSPNs alone failed to suppress locomotion in Pitx3 ak/ak mice, whereas it reliably reduced movement in controls (Fig. 7 e, 7 f). These findings suggest that the loss of ALDH1A1⁺ SNc DANs in Pitx3 ak/ak mice abolishes the suppressive function of patchy dSPNs and instead enhances the locomotion-promoting influence of patchy iSPNs, leading to a paradoxical locomotor enhancement upon patchy SPN activation. This functional reversal likely contributes to the hyperactivity phenotype observed in Pitx3 ak/ak mice. Discussion By integrating molecular mapping, anatomical tracing, and optogenetic manipulation, we reveal a previously unrecognized form of developmental circuit plasticity in the dorsal striatum of Pitx3 ak/ak mice triggered by the selective loss of ALDH1A1⁺ SNc DANs, a subpopulation preferentially vulnerable in PD [ 8 , 13 ]. Our findings demonstrate a selective reorganization of patchy SPNs, marked by a pronounced reduction in the ratio of patchy dSPNs to patchy iSPNs and a reversal of their net influence on motor output. These results suggest that early dopaminergic depletion drives enduring structural and functional adaptations in basal ganglia circuits, which may contribute to altered motor behaviors. Pitx3 ak/ak mice exhibit a profound loss of ALDH1A1⁺ SNc DANs during development [ 18 , 48 ]; paradoxically, however, they display hyperlocomotion [ 19 , 49 ], in stark contrast to the bradykinesia observed following ablation or inhibition ALDH1A1⁺ SNc DAN in adult animals [ 12 , 50 ]. This discrepancy suggests that developmental dopamine loss may engage compensatory mechanisms within the striatum. While prior studies have primarily focused on morphological and electrophysiological alterations in SPNs [ 49 , 51 – 53 ], the specific reorganization of patchy SPNs has not been well characterized. Given that patchy dSPNs form reciprocal connections with ALDH1A1⁺ SNc DANs, the developmental loss of this DAN subtype likely leads to a reduction in patchy dSPNs due to diminished trophic support. Consistent with this notion, we observed a marked decrease in the number of patchy dSPNs in the dorsal striatum and a corresponding loss of their projections to the SNr. Unexpectedly, we also detected a selective increase in patchy iSPNs, resulting in a reversal of the patchy dSPN:iSPN ratio from greater than 1 in controls to less than 1 in Pitx3 ak/ak mutants. This shift likely reflects dopamine-dependent regulation of SPN subtype differentiation, survival, or connectivity during early striatal circuit assembly [ 51 , 54 , 55 ]. The molecular mechanisms underlying the altered SPN composition remain to be elucidated. Whether these changes arise from fate specification or differential survival is unclear. Single-cell transcriptomic profiling of SPNs in Pitx3 ak/ak mice may help define subtype-specific gene expression changes in response to developmental dopamine loss. Anatomical tracing in multiple reporter lines confirmed that the shift in patchy SPN subtype ratio is accompanied by altered projection patterns. In Kremen1 2A − Cre ; Ai14 mice, Pitx3 ak/ak mutants exhibited enhanced projections from patchy SPNs to the GPe and reduced projections to the SNr. These findings were replicated in the Nr4a1 -eGFP reporter line, supporting the conclusion that the patchy iSPN population is selectively expanded and disproportionately contributes to GPe innervation. Further analyses using Pdyn IRES−Cre ; Ai14 and Nr4a1 -eGFP; Drd1 -tdT mice confirmed that patchy dSPNs selectively lost their projections to SNr, with no significant collateralization to GPe. These projection alterations support a model (Fig. 8 ), in which early developmental dopamine loss leads to a reorganization of SPN connectivity that favors indirect pathway output from patch compartments. Optogenetic stimulation provided strong functional validation of the anatomical findings in Pitx3 ak/ak mice. In control mice, activation of patchy SPNs, especially dSPNs, suppressed locomotor activity, consistent with their net inhibitory role on ALDH1A1⁺ DANs and downstream motor circuits [ 33 , 34 , 47 ]. However, in Pitx3 ak/ak mice, the same stimulation paradigm increased locomotor speed, suggesting a functional reversal of patchy SPN output. Moreover, selective activation of patchy dSPNs alone failed to suppress movement in Pitx3 ak/ak mice, reinforcing the idea that these cells are, reduced in number, or have lost effective downstream connectivity. Meanwhile, the enhanced locomotion observed during activation of patchy iSPNs aligns with their increased proportion and GPe innervation. Together, these findings indicate that the hyperactivity observed in Pitx3 ak/ak mice likely stems from an imbalance in patchy SPN circuitry, skewed toward indirect pathway dominance. Most prior studies have attributed the locomotor effects of patchy SPNs to their modulation of DAN activity, particularly ALDH1A1 + SNc DANs [ 33 , 34 , 47 ]. In the absence of these DANs, as in Pitx3 ak/ak mice, how might reorganized patchy SPNs regulate locomotion? An early study using an independent reporter line demonstrated that patchy iSPNs preferentially target a central subregion of the GPe [ 47 ], which corresponds to a distinct subpopulation of GPe neurons associated with arkypallidal (Arky) cells, commonly marked by Npas1 or FoxP2 [ 56 – 58 ]. Unlike prototypical GPe neurons that engage the indirect pathway via the subthalamus nuclei, Arky neurons project back to the striatum, where they provide potent inhibition to SPNs [ 59 – 61 ]. The observed increase in patchy iSPN projections to the GPe in Pitx3 ak/ak mice may result in enhanced inhibition of Arky neurons, thereby reducing their inhibitory feedback to striatal SPNs, including matrix dSPNs (Fig. 8 ). This disinhibition of matrix dSPNs may contribute to the hyperlocomotion observed in Pitx3 ak/ak mice, particularly under conditions of increased patchy iSPN activity. Future studies employing electrophysiological recordings and circuit-mapping approaches will be essential to delineate how patchy iSPNs modulate Arky neuron activity in the GPe of Pitx3 ak/ak mice. Although PD is defined by progressive dopaminergic degeneration, compensatory remodeling of downstream circuits may occur, particularly in early or preclinical stages. The observed increase in patchy iSPN activity may reflect an adaptive mechanism aimed at sustaining motor output in the context of dopamine loss. Despite these important insights, several limitations should be noted. First, the loss of ALDH1A1⁺ SNc DAN in Pitx3 ak/ak mice occurs during early postnatal development. Thus, the circuit adaptations observed in this model likely reflect developmental plasticity, which may differ fundamentally from the compensatory changes that emerge in adult-onset models or in human PD. Whether similar circuit remodeling occurs in progressive PD models or in patients remains to be determined. Second, although Pitx3 ak/ak mice display hyperactivity in open-field tests, they exhibit deficits in motor skill learning [ 53 , 62 ], suggesting that compensatory changes in the striatum are insufficient to fully restore complex motor functions. Third, our analysis was limited to anatomical projections, optogenetics manipulation and behavioral output. Future studies incorporating electrophysiological and neurochemical approaches will be critical to elucidate the functional consequences of these circuit alterations in the dorsal striatum and GPe. Conclusion We identify a novel form of striatal circuit remodeling following the developmental loss of ALDH1A1⁺ SNc DANs in mice. This reorganization selectively alters the balance of patchy SPNs, resulting in a shift toward indirect pathway dominance and functional reversal in their motor output. These findings provide mechanistic insight into how early dopamine depletion can reshape basal ganglia circuitry and highlight patchy iSPNs as potential targets for therapeutic intervention in PD. Abbreviations PD – Parkinson’s disease DANs – Dopaminergic neurons SNc – Substantia nigra pars compacta VTA – Ventral tegmental area ALDH1A1 – Aldehyde dehydrogenase 1 family member A1 SPNs – Striatal projection neurons dSPNs – Direct-pathway striatal projection neurons iSPNs – Indirect-pathway striatal projection neurons GPe – Globus pallidus externa GPi – Globus pallidus interna SNr – Substantia nigra pars reticulata AAV – Adeno-associated virus ChR2 – Channelrhodopsin-2 tdT – tdTomato fluorescent protein TH – Tyrosine hydroxylase CTIP2 – COUP-TF-interacting protein 2 EYFP – Enhanced yellow fluorescent protein Drd1 – Dopamine receptor D1 Drd2 – Dopamine receptor D2 Declarations Consent for publication All authors agree with the content for publication. Availability of data and materials All data generated or analyzed during this study are included in this published article. Source data are provided with this paper. Competing interests The authors declare no competing interests. Funding This research was supported in part by the Intramural Research Program of the National Institutes of Health (H.C., ZIAAG000928, 000959, 000945), the Rodent Behavioral Core of Intramural Research Program of National Institute of Mental Health (MH002952), the National Natural Science Foundation of China (W.D.L., 32220103006; J.J., 32271173), the Natural Science Foundation of Beijing Municipality (J.J., 7242214), and Fujian Provincial Natural Science Foundation (B.S., 2023J01301). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Authors' contributions H.C. conceived and designed the study, wrote the manuscript, and prepared figures with input from all authors. J.D. designed and conducted stereotactic surgeries, optogenetics, histology, behavioral experiments, data analyses, prepared figures, and contributed to writing the methods, figure legends, and manuscript. B.T.S., B.S. and S.L. performed RNAscope in situ hybridization, data analyses, histology, mouse breeding, and behavioral testing. V.M. contributed to RNAscope image analysis, mouse breeding, and histology. L.W. performed optogenetic experiments and contributed to manuscript editing. J.K. conducted image analyses. L.T. assisted with surgeries. L.S. contributed to histological analyses. L.C. performed stereotactic surgeries. J.H.D. contributed to RNA sequencing data analysis. W.D.L. provided guidance on data interpretation and manuscript editing. J.J. made the initial observation on anatomical alterations and contributed to manuscript revision. 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\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePitx3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative image showing ALDH1A1 (magenta), TH (green), and DAPI (blue) immunolabeling in the midbrain of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice. Dashed lines outline the \u003cem\u003esubstantia nigra pars compacta\u003c/em\u003e (SNc) and ventral tegmental area (VTA). Scale bar, 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003ePercentage of ALDH1A1⁺ cells among TH⁺ cells in the SNc and VTA. Unpaired two-tailed t test, SNc: t(6) = 11.23, ****p \u0026lt; 0.0001; VTA: t(6) = 1.989, p = 0.094; n = 4 mice per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Quantification of ALDH1A1⁺ and TH⁺ signal as a percentage of total striatal area across multiple bregma levels (n = 4 mice per genotype).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Group averages of the percentage of ALDH1A1⁺ and TH⁺ areas relative to the total striatum area from (\u003cstrong\u003ed\u003c/strong\u003e). TH: +/ak, 97.76 ± 0.53%; ak/ak, 50.34 ± 3.21%; unpaired two-tailed t-test, t(6) = 14.57, ****p \u0026lt; 0.0001. ALDH1A1: +/ak, 42.20 ± 0.61%; ak/ak, 4.00 ± 0.27%; t(6) = 57.21, ****p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e Locomotor velocity over time during a 30-min open-field test. Two-way ANOVA: time effect, F(2.42, 53.27) = 75.72, ****p \u0026lt; 0.0001; genotype effect, F(1, 22) = 13.02, **p = 0.0016; interaction, F(5, 110) = 28.59, ****p \u0026lt; 0.0001. Multiple comparisons between genotypes: 5 min, ***p = 0.0003; 10 min, *p = 0.042.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg \u003c/strong\u003eAverage velocity across the 30-min session from (f). Unpaired two-tailed t-test, t(22) = 3.608, **p = 0.0016.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e Time spent in surround and center zones of the open field. Unpaired t test, t(22) = 5.174, ****p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei\u003c/strong\u003e Distance traveled in the surround and center zones. Surround: t(22) = 4.817, ****p \u0026lt; 0.0001; center: t(22) = 3.639, **p = 0.0014.\u003c/p\u003e\n\u003cp\u003eSample sizes: \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e, n = 7 males, 6 females; \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e, n = 4 males, 7 females (\u003cstrong\u003ef\u003c/strong\u003e–\u003cstrong\u003ei\u003c/strong\u003e). Data are presented as mean ± SEM. ****p \u0026lt; 0.0001; ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig1PathoBeh.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/53f3b86e5bc813154379de72.png"},{"id":91367520,"identity":"c1f5cfc2-d930-417c-9b58-d900b082f440","added_by":"auto","created_at":"2025-09-15 17:56:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3871583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered numbers of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKremen1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003edSPNs and iSPNs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePitx3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b\u003c/strong\u003e Representative confocal images showing RNAscope labeling of \u003cem\u003eKremen1\u003c/em\u003e(green), \u003cem\u003eDrd1 \u003c/em\u003e(blue), \u0026nbsp;\u003cem\u003eDrd2\u003c/em\u003e (red), and DAPI (gray) in the dorsal striatum (dStr) of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. Right panels show high-magnification views of boxed regions from the left panels. Scale bars: 500 μm (left), 50 μm (right).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Quantification of total dStr area. Unpaired two-tailed t-test: t(4) = 3.239, *p = 0.032.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed, e\u003c/strong\u003e Quantification of spatial patch area (d) and patch number (e) in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e Total numbers of dSPNs and iSPNs in the dStr. Unpaired t test, dSPNs: t(4) = 1.993, p = 0.12; iSPNs: t(4) = 1.845, p = 0.14.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e Numbers of \u003cem\u003eKremen1\u003c/em\u003e⁺ dSPNs and iSPNs in the dStr. \u003cem\u003eKremen1\u003c/em\u003e⁺ dSPNs: t(4) = 12.56, **p = 0.0002; \u003cem\u003eKremen1\u003c/em\u003e⁺ iSPNs: t(4) = 0.87, p = 0.43.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e Proportion of \u003cem\u003eKremen1\u003c/em\u003e⁺ dSPNs among total dSPNs, and \u003cem\u003eKremen1\u003c/em\u003e⁺ iSPNs among total iSPNs. dSPN ratio: t(4) = 8.835, ***p = 0.0009; iSPN ratio: t(4) = 3.187, *p = 0.033.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei \u003c/strong\u003eRatio of dSPNs to iSPNs and of \u003cem\u003eKremen1\u003c/em\u003e⁺ dSPNs to \u003cem\u003eKremen1\u003c/em\u003e⁺ iSPNs. dSPN/iSPN ratio: t(4) = 0.68, p = 0.54; \u003cem\u003eKremen1\u003c/em\u003e⁺ dSPN/iSPN ratio: t(4) = 7.992, **p = 0.0013.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM. n = 3–5 mice per group. ***p \u0026lt; 0.001; **p \u0026lt; 0.01; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig2RNAscope.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/0264f238157a40b88bd960bb.png"},{"id":91367521,"identity":"065cd394-33b1-48c6-a0c2-ae90478f1793","added_by":"auto","created_at":"2025-09-15 17:56:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4417549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered numbers of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNr4a1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-eGFP\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003edSPNs and iSPNs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePitx3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative coronal sections of the dorsal striatum from \u003cem\u003eNr4a1\u003c/em\u003e-eGFP; \u003cem\u003eDrd1\u003c/em\u003e-tdT mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e 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\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Quantification of average numbers of dSPNs and iSPNs in the hemi-dorsal striatum of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice. Unpaired t test, n = 3 mice per each group; dSPNs: t(4) = 1.514, p = 0.2046; iSPNs: t(4) = 1.434, p = 0.2249.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec \u003c/strong\u003eQuantification of average numbers of \u003cem\u003eNr4a1\u003c/em\u003e-eGFP\u003csup\u003e+ \u003c/sup\u003edSPNs and iSPNs in the hemi-dorsal striatum. Unpaired t test, n = 3 mice per each group; dSPNs: t(4) = 4.450, *p = 0.0112; iSPNs: t(4) = 2.185, p = 0.0942.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Percentage of \u003cem\u003eNr4a1\u003c/em\u003e-eGFP\u003csup\u003e+ \u003c/sup\u003eSPNs among total SPNs in the dorsal striatum. Unpaired t test, n = 3 mice per each group; dSPNs: t(4) = 3.978, *p = 0.0164; iSPNs: t(4) = 4.063, *p = 0.0153.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Ratio of dSPNs to iSPNs and \u003cem\u003eNr4a1\u003c/em\u003e-eGFP\u003csup\u003e+ \u003c/sup\u003edSPNs to\u003cem\u003e Nr4a1\u003c/em\u003e-eGFP\u003csup\u003e+ \u003c/sup\u003eiSPNs in the dorsal striatum. Unpaired t test, n = 3 mice per each group; dSPNs/iSPNs: t(4) =2.07, p = 0.1073; \u003cem\u003eNr4a1\u003c/em\u003e-eGFP\u003csup\u003e+ \u003c/sup\u003edSPNs/\u003cem\u003e Nr4a1\u003c/em\u003e-eGFP\u003csup\u003e+ \u003c/sup\u003eiSPNs: t(4) =14.03, ***p = 0.0001. Data are presented as mean ± SEM. n = 3 mice per group. ***p \u0026lt; 0.001; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig3Nr4a1GFPD1tdT.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/e348bf67c80e5c1e186a5b10.png"},{"id":91368020,"identity":"e794028f-0909-448c-bc39-458b8c2188e6","added_by":"auto","created_at":"2025-09-15 18:04:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7041321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered distribution and projection patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKremen1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e⁺ SPNs in the dorsal striatum of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePitx3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative sagittal brain sections from \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e2A-Cre\u003c/sup\u003e; Ai14 mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Coronal sections showing altered tdT signal distribution in the dorsal striatum of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. Right panel shows a magnified view of the dashed region in the left panel. Arrow points to the dispersed tdT\u003csup\u003e+\u003c/sup\u003e neurons in DLS. Scale bar, 500 μm. Abbreviations: DLS, dorsolateral striatum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Representative coronal sections showing increased tdT signal in the dorsal GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. Right panel shows a magnified view of the dashed area. Scale bar, 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Reduced tdT signal in the SNr of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. Arrows indicate dendron-bouquet structures. Scale bar, 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Quantification of average tdT signal intensity and the percentage of tdT-positive area in the GPe. Unpaired t test, n = 3 mice per genotype. Signal intensity: t(4) = 2.818, *p = 0.0479; positive area: t(4) = 4.082, *p = 0.0151.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e Quantification of average tdT signal intensity and the percentage of tdT-positive area in the SNr. Unpaired t test, n = 3 mice per genotype. Signal intensity: t(4) = 3.231, *p =0.0319; positive area: t(4) = 4.129, *p = 0.0145.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM. *p \u0026lt; 0.05 indicates statistical significance.\u003c/p\u003e","description":"","filename":"Fig4Ai14.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/ed6dad62d14c10db8b48a6e5.png"},{"id":91368604,"identity":"012750e5-c9f6-48b9-821f-9994a61b6c5a","added_by":"auto","created_at":"2025-09-15 18:12:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7825845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered distribution and projection patterns of patchy SPNs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNr4a1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-eGFP mice on a \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePix3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e background.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative coronal sections from rostral to caudal levels of \u003cem\u003eNr4a1\u003c/em\u003e-eGFP mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e backgrounds, stained for TH (magenta). Note the altered GFP signal distribution in the dorsal striatum and dorsal GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. Scale bar, 1 mm. Abbreviations: dStr, dorsal striatum; GPe, globus pallidus externus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e GFP signal distribution in the GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice merged with PV (red) expression in the GPe. Dotted lines outline the GPe. Solid lines indicate the regions used for intensity analysis in \u003cstrong\u003ed\u003c/strong\u003e. Scale bar, 250 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Quantification of average GFP signal intensity in the GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (n = 3 mice per genotype). Unpaired t test, t(4) = 3.465, *p = 0.0257.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Intensity profile analysis of GFP signals along the solid lines in \u003cstrong\u003eb\u003c/strong\u003e. GFP signal intensity is higher in the dorsal GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (magenta) compared to the controls (gray).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Colocalization analysis of GFP and PV expression in the GPe of\u003cem\u003e Pitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice (n = 3 mice). M1 = 0.97 ± 0.0007; M2 = 0.92 ± 0.04. M1: the proportion of PV signal overlapping with GFP.\u0026nbsp;M2: the proportion of GFP signal overlapping with PV.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e Representative coronal SNr sections from \u003cem\u003eNr4a1\u003c/em\u003e-eGFP mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e backgrounds, stained for TH (magenta) and DRD1 (red). DRD1 was used to outline the SNr. Arrows indicate dendron-bouquet structure in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak \u003c/sup\u003emice; arrowhead indicates reduced GFP signal in the ventral SNr. Solid lines indicate the regions used for intensity analysis in \u003cstrong\u003eg.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg \u003c/strong\u003eIntensity profile analysis of GFP signals along the solid lines in the SNr from \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e (top panel) and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e (bottom panel) mice in \u003cstrong\u003ef\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh\u003c/strong\u003e Percentage of GFP-expressing area within the SNr (Drd1\u003csup\u003e+\u003c/sup\u003e area) in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (n = 3 mice per each genotype). Unpaired t test, t(4) = 9.578, ***p = 0.0007.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg\u003c/strong\u003e Quantification of average GFP signal intensity in the SNr of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (n = 3 mice per genotype). Unpaired t test, t(4) = 0.07197, p = 0.9461.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM. ***p \u0026lt; 0.001; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig5Pitx3Nr4a1GFP.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/598681f801a4b6eca8441d9f.png"},{"id":91367523,"identity":"eb78cf2e-08ab-46ea-85be-71c3f9b7dc20","added_by":"auto","created_at":"2025-09-15 17:56:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4440708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered distribution and projection patterns of patchy SPNs in the dorsal striatum of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePdyn\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eIRES-Cre\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e; Ai14 mice on a \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePix3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e background.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative coronal sections from rostral to caudal levels of \u003cem\u003ePdyn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eIRES\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003eCre\u003c/sup\u003e; Ai14 mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e backgrounds, stained for TH (green) and DAPI (blue). Scale bar, 1 mm. Abbreviations: dStr, dorsal striatum; GPe, globus pallidus externus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Representative coronal SNr sections from \u003cem\u003ePdyn\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003csup\u003eIRES\u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003csup\u003eCre\u003c/sup\u003e; Ai14 mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e backgrounds, stained for TH (magenta) and DAPI (blue). Arrows indicate dendron-bouquet structures in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak \u003c/sup\u003emice. Scale bar, 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Quantification of average tdT signal intensity in the GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (n = 3 mice per genotype). Unpaired t test, t(4) = 0.26, p = 0.8077.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Quantification of average tdT signal intensity in the SNr of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (n = 3 mice per genotype). Unpaired t test, t(4) = 3.51, *p = 0.0247.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Percentage of tdT-expressing area within the SNr in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (n = 3 mice per each genotype). Unpaired t test, t(4) = 5.406, **p = 0.0057.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SEM. **P\u0026lt;0.01; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig6Pitx3pdynAi14.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/ee10c2d9c33262bea0d60544.png"},{"id":91368019,"identity":"23cd20b0-597f-465c-a1a6-c4173ea5a5f6","added_by":"auto","created_at":"2025-09-15 18:04:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1872709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptogenetic activation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKremen1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e SPNs in the dorsal striatum promote locomotion in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePitx3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eSchematic illustrating AAV-FLEX-ChR2 vector injection and optical fiber implantation for selective activation of \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e SPNs in the dorsal striatum or the SN in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e Instantaneous velocity aligned to optogenetics stimulations (10s, blue shaded area) of \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e SPNs in the dorsal striatum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed\u003c/strong\u003e Average velocity during pre-stimulation (Pre), stimulation (Stim, blue shaded area), and post-stimulation (Post) in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak \u003c/sup\u003emice (left) and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (right) from panel \u003cstrong\u003ec\u003c/strong\u003e. For\u003cem\u003e Pitx3\u003c/em\u003e\u003csup\u003e+/ak \u003c/sup\u003emice: \u0026nbsp;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 \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e 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. \u0026nbsp;Error bars represent mean ± SEM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee\u003c/strong\u003e Instantaneous velocity aligned to optogenetics stimulations (10s, blue shaded area) of \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e dSPNs in the SN.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef\u003c/strong\u003e Average velocity during pre-stimulation (Pre), stimulation (Stim, blue shaded area), and post-stimulation (Post) in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak \u003c/sup\u003emice (left) and \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice (right) from panel \u003cstrong\u003ee\u003c/strong\u003e. For\u003cem\u003e Pitx3\u003c/em\u003e\u003csup\u003e+/ak \u003c/sup\u003emice: \u0026nbsp;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 \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e 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.\u003c/p\u003e","description":"","filename":"Fig7ChR2.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/89ba0457bce2b78513bc001d.png"},{"id":91367527,"identity":"e90044be-dc4d-4457-8401-d333f82409c6","added_by":"auto","created_at":"2025-09-15 17:56:32","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":756154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of patchy SPN reorganization in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePitx3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eak/ak \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis schematic illustrates a selective reorganization of patchy SPNs in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice, 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 \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak \u003c/sup\u003emice, particularly under conditions of heightened patchy iSPN activity.\u003c/p\u003e","description":"","filename":"Fig8ModelGPe.png","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/4ad8858d8f24d815479e2bce.png"},{"id":99545337,"identity":"fd297f9f-a620-4e4b-995f-0b62675ebfaf","added_by":"auto","created_at":"2026-01-05 16:06:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33996624,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/e1eea5d9-cba8-48a6-ae59-a0c8bb787a5d.pdf"},{"id":91368025,"identity":"49fb8431-a531-4672-95b4-0bd97a8b2326","added_by":"auto","created_at":"2025-09-15 18:04:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6803049,"visible":true,"origin":"","legend":"","description":"","filename":"Supp20250818.docx","url":"https://assets-eu.researchsquare.com/files/rs-7401124/v1/88306ca6f654931a8adbea51.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Developmental Dopamine Loss Rewires Striatal Circuits to Promote Locomotion","fulltext":[{"header":"Background","content":"\u003cp\u003ePatients with Parkinson\u0026rsquo;s disease (PD) experience progressive motor symptoms, including resting tremor, slowed movement, and impaired posture and balance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In addition to these motor issues, they often suffer from non-motor symptoms such as depression and dementia [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While medications and surgical interventions can improve motor function, long-term medication can lead to severe side effects, including dyskinesia and impulse control disorders [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, there is a continuing need for new mechanistic insights and therapeutic strategies to improve treatment options for the growing number of PD patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe motor symptoms of PD are primarily associated with the degeneration of midbrain dopaminergic neurons (DANs) in the \u003cem\u003esubstantia nigra compacta\u003c/em\u003e (SNc) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], particularly the Aldehyde Dehydrogenase 1A1-positive (ALDH1A1\u003csup\u003e+\u003c/sup\u003e) subset in its ventral tier [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The ALDH1A1\u003csup\u003e+\u003c/sup\u003e SNc DANs accounts for 60\u0026ndash;70% DANs in human and rodent SNc and mainly project to the dorsal portion of dorsal striatum [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In naturally occurring Pituitary homeobox 3 (\u003cem\u003ePitx3\u003c/em\u003e)-deficient aphakia (or \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e) mice, midbrain DAN differentiation remains unaffected at embryonic day 11.5 (E11.5) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, a noticeable reduction in tyrosine hydroxylase (TH)-positive cells destined for the SNc emerges by E12.5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], leading to a significant loss of SNc DANs by postnatal day 3 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, DANs in the ventral tegmental area (VTA) remain largely unaffected [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Notably, the depleted SNc DANs in these mice predominantly belong to the ALDH1A1\u003csup\u003e+\u003c/sup\u003e DAN subpopulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, while acute genetic ablation of ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs in adult mice slows locomotion [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the developmental loss of these neurons in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice does not decrease nighttime movement [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Instead, it increases daytime activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This adaptation likely involves striatal projection neurons (SPNs) in the dorsal striatum, which are the primary targets of midbrain DAN input [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Investigating these compensatory mechanisms could provide valuable insights into neuromodulatory strategies for addressing PD-related motor deficits.\u003c/p\u003e\u003cp\u003eIn the dorsal striatum, SPNs are broadly classified into two major subtypes based on their molecular identity and projection targets: direct-pathway SPNs (dSPNs), which express dopamine receptor D1 (DRD1) and project to the internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr), and indirect-pathway SPNs (iSPNs), which express dopamine receptor D2 (DRD2) and project to the external globus pallidus (GPe) [\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Both dSPNs and iSPNs are distributed across two complementary compartments\u0026mdash;the patch (or striosome) and the matrix, which differ in anatomical organization, gene expression profiles, and connectivity patterns [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, SPNs with molecular characteristics of patch neurons can also be found scattered within the matrix compartment; these are referred to as \"exo-patch\" SPNs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Since molecularly labeling does not distinguish between canonical patch and exo-patch locations, we use the term \u0026ldquo;patchy SPNs\u0026rdquo; to collectively refer to both populations in this study. Among midbrain DANs, ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs in the SNc receive the strongest monosynaptic inhibitory input from patchy dSPNs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Recent studies have shown that activation of patchy dSPNs suppresses locomotion via inhibition of ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs and reduction of dopamine release [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], whereas patchy iSPNs appear to facilitate movement, highlighting compartment- and cell type-specific functional roles [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, it remains unclear how the developmental loss of ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice affects the organization and function of SPNs, particularly patchy SPNs.\u003c/p\u003e\u003cp\u003eIn this study, we examine the structural and functional reorganization of SPNs, with a focus on patchy SPNs, in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. We analyze the composition and distribution of molecularly defined patchy dSPNs and iSPNs in the dorsal striatum and assess their projection patterns to the GPe and SNr. Additionally, we use optogenetic approaches to selectively activate patchy dSPNs and iSPNs to determine their impact on locomotion.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e All mouse procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Intramural Research Program at the National Institute on Aging (NIA), NIH, and conducted in accordance with institutional and NIH guidelines. All mouse lines were maintained as heterozygotes on a C57BL/6J background. The following strains were obtained from The Jackson Laboratory: \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e (Stock No: 000942) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eDrd1\u0026ndash;\u003c/em\u003etdTomato (Stock No: 016204) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], \u003cem\u003ePdyn\u003c/em\u003e\u003csup\u003eIRES\u0026minus;Cre\u003c/sup\u003e (Stock No: 027958) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and Ai14 (Stock No: 007908) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice were backcrossed with C57BL/6J for more than five generations. The \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e2A\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e knock-in mice [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] were generated by Shanghai Model Organisms Inc. (Shanghai, China), while the \u003cem\u003eNr4a1\u0026ndash;\u003c/em\u003eeGFP (Stock No: 036737-UCD) transgenic mice [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] were obtained from the Mutant Mouse Resource \u0026amp; Research Centers (MMRRC).\u003c/p\u003e\u003cp\u003eTo examine the projections of patchy dSPNs and iSPNs, three patchy SPN mouse lines (\u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e2A\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e, \u003cem\u003ePdyn\u003c/em\u003e\u003csup\u003eIRES\u0026minus;Cre\u003c/sup\u003e, and \u003cem\u003eNr4a1\u0026ndash;\u003c/em\u003eeGFP) were bred into the \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e background, and their littermates \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e were used as control in the experiments. Both male and female mice were used in all experiments. Mice were group-housed (2\u0026ndash;5 per cage) under a 12-hour light/dark cycle with ad libitum access to water and standard chow. Behavioral experiments were conducted during the light phase. Littermates were randomly assigned to experimental groups before study onset.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA\u003c/b\u003e \u003cb\u003eIn Situ\u003c/b\u003e \u003cb\u003eHybridization and Image Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA \u003cem\u003ein situ\u003c/em\u003e hybridization (RNAscope, ACDBio) was performed to detect \u003cem\u003eDrd1\u003c/em\u003e, \u003cem\u003eDrd2\u003c/em\u003e, and \u003cem\u003eKremen1\u003c/em\u003e mRNAs in the dorsal striatum of adult C57BL/6J mice. Mice were euthanized with CO₂ inhalation. Brains were fresh-frozen on dry ice and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Coronal sections (12 \u0026micro;m) were cut on a cryostat (Leica Biosystems) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\u003cp\u003eRNAscope was performed using the Multiplex Fluorescent Reagent Kit v2 per the manufacturer's instruction. Probes used included: \u003cem\u003eDrd1\u003c/em\u003e (Cat. No. #401901), \u003cem\u003eDrd2\u003c/em\u003e (Cat. No. #406501), and \u003cem\u003eKremen1\u003c/em\u003e (Cat. No. #425771). Images were acquired on a Zeiss LSM 780 laser scanning confocal microscope with 20\u0026times; or 40\u0026times; objectives. Five sections were analyzed spanning a rostro-caudal range from approximately 1.34 mm to \u0026minus;\u0026thinsp;0.34 mm relative to Bregma.\u003c/p\u003e\u003cp\u003eImage analysis was conducted using Imaris v10.0 (Bitplane, Belfast, UK). Dorsal and ventral striatal regions were delineated using the Allen Brain Atlas. Spatial patches were defined as clusters of at least five \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e SPNs with a minimum density of 200 cells/mm\u0026sup2;. Channels (\u003cem\u003eDrd1\u003c/em\u003e, \u003cem\u003eDrd2\u003c/em\u003e, \u003cem\u003eKremen1\u003c/em\u003e, and DAPI) were analyzed with consistent parameters within each batch; minor adjustments across batches ensured optimal quantification. DAPI\u003csup\u003e+\u003c/sup\u003e cells were classified as \u003cem\u003eDrd1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e (dSPNs) or \u003cem\u003eDrd2\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e (iSPNs) based on mean fluorescence intensity. Overlap with \u003cem\u003eKremen1\u003c/em\u003e signal was used to quantify patchy dSPNs and iSPNs. Data points represent averages across multiple bilateral striatal sections.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eMice were anesthetized with pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed overnight at 4\u0026deg;C, then cryoprotected in 30% sucrose (PBS-buffered) for \u0026ge;\u0026thinsp;48 hours. Coronal sections (40 \u0026micro;m) were cut and stored in PBS with 4% sodium azide at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003eSections were blocked for 1 hour at room temperature in 10% normal donkey serum, 0.5% BSA, and 0.3% Triton-X-100. They were incubated with primary antibodies overnight or for 48 hours at 4\u0026deg;C, washed (3 \u0026times; 10 min, PBS), and then incubated with secondary antibodies for 1 hour. Some sections were counterstained with DAPI (0.5 mg/mL, 1 min, Invitrogen, D1306) and mounted using ProLong Gold Antifade Mountant (Life Technologies). Images were captured using a Zeiss LSM 780 or LSM 980 confocal microscope.\u003c/p\u003e\u003cp\u003ePrimary antibodies used: polyclonal rabbit anti-PITX3 (1:500) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], rabbit anti-TH (Pel-Freez Biologicals, P40101; 1:1000), goat anti-ALDH1A1 (R\u0026amp;D system; 1:1000), guinea pig anti-PV (Swant, GP72; dilution 1:1000), goat anti-ChAT (AB144P, Millipore; dilution 1:500) and rat anti-CTIP2 (abcam, ab18465, 1:500). Secondary antibodies (Life Technologies) were selected for appropriate fluorophore spectra.\u003c/p\u003e\n\u003ch3\u003eQuantification of TH and ALDH1A1 Neurons\u003c/h3\u003e\n\u003cp\u003eMidbrain sections (40 \u0026micro;m) spanning Bregma \u0026minus;\u0026thinsp;2.70 mm to \u0026minus;\u0026thinsp;3.80 mm (seven sections per mouse) were stained for TH, ALDH1A1 and DAPI. Z-stack confocal images were captured at 20\u0026times; magnification. Neurons in the SNc and VTA were quantified using the \u0026ldquo;Detect Cells\u003cem\u003e\u0026rdquo;\u003c/em\u003e functions in NeuroInfo (MBF Bioscience). Regions were manually delineated using previously established anatomical landmarks. Total neuron numbers were estimated using \u0026ldquo;Area Under the Curve\u0026rdquo; analysis in GraphPad Prism 10.\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuantification of TH\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eand ALDH1A1\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eAxon Terminal in the Striatum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFive to six striatal sections across a rostro-caudal series (approximately 1.42mm to -0.2 mm relative to Bregma) were co-stained for TH and ALDH1A1. Images were acquired with 10 \u0026times; objective on the Zeiss LSM780. ALDH1A1\u003csup\u003e+\u003c/sup\u003e areas in the dorsal striatum were outlined using intensity threshold in ImageJ. For each section, the percentage of ALDH1A1\u003csup\u003e+\u003c/sup\u003e area relative to total striatal area was calculated.\u003c/p\u003e\n\u003ch3\u003eQuantification of Nr4a1-eGFP cells in the dorsal striatum\u003c/h3\u003e\n\u003cp\u003eIn \u003cem\u003eNr4a1\u003c/em\u003e-eGFP; \u003cem\u003eDrd1\u003c/em\u003e-tdTomato mice on either \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e or \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e backgrounds, striatal sections spanning from Bregma 1.34mm to -0.34mm (5 sections per each mouse) were stained for CTIP2 and imaged using a Zeiss LSM 780 laser scanning confocal microscope with a 10\u0026times; objective. Dorsal striatal regions were delineated according to the Allen Brain Atlas. Cell quantification was performed using Imaris v10.0 (Bitplane, Belfast, UK). CTIP2\u003csup\u003e+\u003c/sup\u003e cells were identified as SPNs and further classified as tdT\u003csup\u003e+\u003c/sup\u003e (dSPNs) and tdT-negative (iSPNs) based on median fluorescence intensity. Overlap with GFP\u003csup\u003e+\u003c/sup\u003e signal was used to quantify Nr4a1-eGFP\u003csup\u003e+\u003c/sup\u003e dSPNs and iSPNs. Minor threshold adjustments were applied across batches to ensure optimal quantification.\u003c/p\u003e\n\u003ch3\u003eQuantification of fluorescence intensity and cell density\u003c/h3\u003e\n\u003cp\u003eFluorescence intensity and cell density were quantified using ImageJ. For each mouse, 3\u0026ndash;8 striatal or midbrain sections at different Bregma levels were analyzed. For SNr and GPe regions, mean RFP or GFP fluorescence intensity was calculated by delineating the regions of interest based on anatomical landmarks in the Allen Brain Atlas.\u003c/p\u003e\u003cp\u003eTo assess fluorescence distribution, \u0026ldquo;Plot Profile\u0026rdquo; function was used to generate intensity plots along the drawn line. Colocalization analysis between GFP\u003csup\u003e+\u003c/sup\u003e and PV\u003csup\u003e+\u003c/sup\u003e signals was performed using the \u0026ldquo;Coloc2\u0026rdquo; function in ImageJ to calculate Manders' coefficients M1 and M2, indicating the proportion of GFP signal overlapping with PV and vice versa.\u003c/p\u003e\u003cp\u003eFor cell density analysis, PV\u003csup\u003e+\u003c/sup\u003e and ChAT\u003csup\u003e+\u003c/sup\u003e cells were automatically counted in the dorsal striatum by setting the particle diameter. The counting area was measured, and cell density was calculated as the number of positive cells per mm\u0026sup2;.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStereotaxic Viral Injection and Optic Fiber Implantation\u003c/h2\u003e\u003cp\u003eStereotaxic surgeries were performed under aseptic conditions. Mice (2\u0026ndash;4 months old) were anesthetized with 1\u0026ndash;2% isoflurane and placed in a stereotaxic frame (Kopf Instruments). A total of 700 nL of AAV was bilaterally injected into the dorsal striatum (AP: +0.9 mm; ML: \u0026plusmn;2.2 mm; DV: \u0026minus;2.5 mm) at 75 nL/min using a microinjector (Stoelting). Vectors included AAV1-EF1a-double floxed-hChR2(H134R)-EYFP (#20298) and AAV1-Ef1a-DIO-EYFP (#27056) from Addgene (Watertown, MA, USA). The needle was left in place for 5 minutes post-injection before withdrawn. Incisions were closed and mice recovered in home cages.\u003c/p\u003e\u003cp\u003eAfter three weeks, optical fibers (200 \u0026micro;m core, 0.39 NA; Thorlabs) were bilaterally implanted targeting either the dorsal striatum (AP: +1.0 mm; ML: \u0026plusmn;1.5 mm; DV: \u0026minus;2.2 mm to \u0026minus;\u0026thinsp;2.7 mm) or SNr (AP: \u0026minus;3.1 mm; ML: \u0026plusmn;1.5 mm; DV: \u0026minus;4.1 mm DV). Fibers were secured with radiopaque adhesive cement (C\u0026amp;B METABOND, Parkell) and incisions were sealed with Vetbond tissue adhesive (3M). Mice recovered for \u0026ge;\u0026thinsp;1 week before testing.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOpen-Field Spontaneous Locomotion\u003c/h3\u003e\n\u003cp\u003eFreely moving mice were assessed for spontaneous locomotion via video tracking. Prior to testing, mice were habituated for 30 minutes. The arena (50 \u0026times; 50 cm, opaque gray) was lit diffusely using an enclosed 20W lamp. Mice were recorded for 30 minutes at 30 Hz from above using a digital camera. For analysis, the arena was divided into a center zone (central 25 \u0026times; 25 cm) and a surrounding zone. Data on velocity, distance, and time traveled were analyzed with EthoVision XT (Noldus). For beam break tests, mice were placed in a 43 \u0026times; 23 cm arena with infrared sensors. Chambers were cleaned with 50% ethanol between trials.\u003c/p\u003e\n\u003ch3\u003eOptogenetics in the Open Field Test\u003c/h3\u003e\n\u003cp\u003eLight delivery was controlled using an LED source and commutator (PlexBright, Plexon) connected via a patch cable (200 \u0026micro;m, 0.39 NA). Connections were made with ceramic sleeves (Thorlabs). Light power (465 nm) was calibrated to 3 mW at the fiber tip (Thorlabs PM100D). For ChR2 activation, 5-ms light pulses were delivered at varying frequencies and durations controlled via a TTL signal generator (OPTG-4, Doric Lenses).\u003c/p\u003e\u003cp\u003eMice were habituated for 30 minutes before testing. The arena (50 \u0026times; 50 cm, transparent walls) was cleaned between sessions. Mice were recorded from both top and side views (Logitech cameras) at 15 Hz. After a 3-minute baseline, optogenetic stimulation was applied bilaterally in 10 sec ON / 1 min OFF cycles. Video and TTL signals were synchronized using Synapse software (TDT). Locomotion metrics were analyzed in EthoVision XT.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed in GraphPad Prism 10 and custom MATLAB scripts (MathWorks). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Specific statistical tests are reported in figure legends. Significance was assessed using two-tailed t-tests and one-way ANOVA, with thresholds of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (\u003csup\u003e*\u003c/sup\u003e), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (\u003csup\u003e**\u003c/sup\u003e), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (\u003csup\u003e***\u003c/sup\u003e), and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (\u003csup\u003e****\u003c/sup\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePitx3\u003c/b\u003e\u003csup\u003e\u003cb\u003eak/ak\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice exhibit hyperlocomotion despite the loss of ALDH1A1⁺ DANs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsistent with previous findings [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], PITX3 expression was completely absent in midbrain DANs of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice (\u003cb\u003eSupplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Loss of PITX3 resulted in a selective depletion of ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs in the ventral SNc, whereas ALDH1A1\u0026ndash; SNc DANs and VTA DANs remained largely intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Accordingly, ALDH1A1\u003csup\u003e+\u003c/sup\u003e axon projections were severely reduced in the dorsal striatum of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnexpectedly, \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice displayed hyperactivity in the open-field test compared to littermate controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Mutant mice also spent significantly more time and traveled longer distances in the peripheral zone relative to the center (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), indicative of elevated anxiety-like behavior. This paradoxical increase in locomotor activity despite the loss of ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs suggests a possible developmental compensatory mechanism, potentially involving circuit-level reorganization of SPNs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSignificant reduction in patchy dSPNs and increase in patchy iSPNs in\u003c/b\u003e \u003cb\u003ePitx3\u003c/b\u003e\u003csup\u003e\u003cb\u003eak/ak\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess changes in SPN subtypes, we performed multiplexed RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization using probes for \u003cem\u003eDrd1\u003c/em\u003e, \u003cem\u003eDrd2\u003c/em\u003e, and \u003cem\u003eKremen1\u003c/em\u003e, which label dSPNs, iSPNs, and a subset of patchy SPNs, respectively [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This approach allowed us to map the composition and distribution of SPN subtypes in the dorsal striatum of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e and littermate control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Although \u003cem\u003ePitx3\u003c/em\u003e-deficiency led to a significant reduction in the overall surface area of the dorsal striatum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), it did not alter the size or number of patch-like structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), nor did it affect the total numbers of dSPNs or iSPNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, the number of \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e patchy dSPNs was substantially reduced in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, whereas the number of patchy iSPNs showed a slight increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Quantification of the proportion of \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells within the total dSPN and iSPN populations revealed opposite trends: the fraction of patchy dSPNs was significantly decreased, while that of patchy iSPNs was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). As a result, the ratios of patchy dSPNs to patchy iSPNs was reversed, shifting from 1.7 in control mice to 0.7 in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003eThese findings were corroborated in an independent patchy SPN reporter model, \u003cem\u003eNr4a1\u003c/em\u003e-eGFP; \u003cem\u003eDrd1\u003c/em\u003e-tdTomato; \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, in which patchy SPNs were labeled by GFP [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], dSPNs by tdTomato (tdT) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and iSPNs identified as CTIP2-positive but tdT-negative (CTIP2\u003csup\u003e+\u003c/sup\u003e /tdT\u003csup\u003e\u0026ndash;\u003c/sup\u003e) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-e). CTIP2 is a general marker for SPNs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. By contrast, we observed no apparent changes in PV\u003csup\u003e+\u003c/sup\u003e and ChAT\u003csup\u003e+\u003c/sup\u003e interneurons in the dorsal striatum of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice (\u003cb\u003eSupplementary Fig. S2\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTogether, these findings from two independent models demonstrate that PITX3 loss selectively alters patchy SPN composition, characterized by a decrease in patchy dSPNs and a relative increase in patchy iSPNs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReduced SNr projections from patchy dSPNs and increased GPe projections from patchy iSPNs in\u003c/b\u003e \u003cb\u003ePitx3\u003c/b\u003e\u003csup\u003e\u003cb\u003eak/ak\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the output pathways of patchy SPNs, we generated \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e2A\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e; Ai14 bigenic mice on \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003e+/ak\u003c/sup\u003e or \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e backgrounds, enabling selective labeling of \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e patchy SPNs with tdT. In \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, axonal projections to the GPe were increased, whereas projections to the SNr were markedly reduced compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Moreover, patchy SPNs in these mice appeared more dispersed and lacked the characteristic clustered organization within the superficial dorsolateral striatum (DLS) (arrow, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The increase in projection was localized primarily to the dorsal GPe (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec); while dendron-bouquet structures, formed by the dendrites of DANs and axons of patchy dSPN [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], were entirely absent in the SNr (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Quantitative analyses of serial GPe and SNr sections confirmed these projection changes (\u003cb\u003eFig, 4e, 4f\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilar alterations in both somatic distribution in the DLS and projection patterns in GPe and SNr were observed in \u003cem\u003eNr4a1\u003c/em\u003e-eGFP; \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-i). Given that dSPNs can send axonal collaterals to the GPe [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], we next examined whether increased dSPN collateralization might account for the enhanced GPe projections. To test this, we analyzed \u003cem\u003ePdyn\u003c/em\u003e \u003csup\u003eIRES\u0026minus;Cre\u003c/sup\u003e; Ai14 mice, in which tdT preferentially labels patchy dSPNs [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In these mice, PITX3 loss did not affect GPe projections but still led to a reduction in SNr projections (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-e). To further investigate the contribution of dSPN collaterals, we examined \u003cem\u003eNr4a1\u003c/em\u003e-eGFP; \u003cem\u003eDrd1\u003c/em\u003e-tdT mice and found no difference in tdT signal intensity in the GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e and control mice (\u003cb\u003eSupplementary Fig. S3\u003c/b\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaken together, these complementary datasets suggest that PITX3 deficiency disrupts the balance of patchy SPN output pathways by reducing SNr projections due to a loss of patchy dSPNs and increasing GPe projections through an expansion of patchy iSPNs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptogenetic stimulation of patchy SPNs reveals reversed motor effects in\u003c/b\u003e \u003cb\u003ePitx3\u003c/b\u003e\u003csup\u003e\u003cb\u003eak/ak\u003c/b\u003e\u003c/sup\u003e \u003cb\u003emice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRecent studies have shown that patchy dSPNs suppress locomotion via inhibition of ALDH1A1\u003csup\u003e+\u003c/sup\u003e DANs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], whereas patchy indirect pathway SPNs (iSPNs) exert a weaker locomotion-promoting effect [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Under normal conditions, the net outcome of patchy SPN activation is locomotion inhibition [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. To examine the functional consequences of patchy SPN reorganization in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, we performed optogenetic stimulation to activate either both patchy dSPNs and iSPNs or dSPNs alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). In control mice, stimulation of both patchy SPN subtypes significantly reduced locomotor velocity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), consistent with the dominant inhibitory role of patchy dSPNs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In contrast, the same stimulation paradigm in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice resulted in a marked increase in locomotor speed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed), indicating a reversal of functional output. Notably, direct stimulation of patchy dSPNs alone failed to suppress locomotion in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, whereas it reliably reduced movement in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). These findings suggest that the loss of ALDH1A1⁺ SNc DANs in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice abolishes the suppressive function of patchy dSPNs and instead enhances the locomotion-promoting influence of patchy iSPNs, leading to a paradoxical locomotor enhancement upon patchy SPN activation. This functional reversal likely contributes to the hyperactivity phenotype observed in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBy integrating molecular mapping, anatomical tracing, and optogenetic manipulation, we reveal a previously unrecognized form of developmental circuit plasticity in the dorsal striatum of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice triggered by the selective loss of ALDH1A1⁺ SNc DANs, a subpopulation preferentially vulnerable in PD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Our findings demonstrate a selective reorganization of patchy SPNs, marked by a pronounced reduction in the ratio of patchy dSPNs to patchy iSPNs and a reversal of their net influence on motor output. These results suggest that early dopaminergic depletion drives enduring structural and functional adaptations in basal ganglia circuits, which may contribute to altered motor behaviors.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice exhibit a profound loss of ALDH1A1⁺ SNc DANs during development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]; paradoxically, however, they display hyperlocomotion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], in stark contrast to the bradykinesia observed following ablation or inhibition ALDH1A1⁺ SNc DAN in adult animals [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This discrepancy suggests that developmental dopamine loss may engage compensatory mechanisms within the striatum. While prior studies have primarily focused on morphological and electrophysiological alterations in SPNs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], the specific reorganization of patchy SPNs has not been well characterized. Given that patchy dSPNs form reciprocal connections with ALDH1A1⁺ SNc DANs, the developmental loss of this DAN subtype likely leads to a reduction in patchy dSPNs due to diminished trophic support. Consistent with this notion, we observed a marked decrease in the number of patchy dSPNs in the dorsal striatum and a corresponding loss of their projections to the SNr. Unexpectedly, we also detected a selective increase in patchy iSPNs, resulting in a reversal of the patchy dSPN:iSPN ratio from greater than 1 in controls to less than 1 in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mutants. This shift likely reflects dopamine-dependent regulation of SPN subtype differentiation, survival, or connectivity during early striatal circuit assembly [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The molecular mechanisms underlying the altered SPN composition remain to be elucidated. Whether these changes arise from fate specification or differential survival is unclear. Single-cell transcriptomic profiling of SPNs in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice may help define subtype-specific gene expression changes in response to developmental dopamine loss.\u003c/p\u003e\u003cp\u003eAnatomical tracing in multiple reporter lines confirmed that the shift in patchy SPN subtype ratio is accompanied by altered projection patterns. In \u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e2A\u0026thinsp;\u0026minus;\u0026thinsp;Cre\u003c/sup\u003e; Ai14 mice, \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mutants exhibited enhanced projections from patchy SPNs to the GPe and reduced projections to the SNr. These findings were replicated in the \u003cem\u003eNr4a1\u003c/em\u003e-eGFP reporter line, supporting the conclusion that the patchy iSPN population is selectively expanded and disproportionately contributes to GPe innervation. Further analyses using \u003cem\u003ePdyn\u003c/em\u003e\u003csup\u003eIRES\u0026minus;Cre\u003c/sup\u003e; Ai14 and \u003cem\u003eNr4a1\u003c/em\u003e-eGFP; \u003cem\u003eDrd1\u003c/em\u003e-tdT mice confirmed that patchy dSPNs selectively lost their projections to SNr, with no significant collateralization to GPe. These projection alterations support a model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), in which early developmental dopamine loss leads to a reorganization of SPN connectivity that favors indirect pathway output from patch compartments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOptogenetic stimulation provided strong functional validation of the anatomical findings in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice. In control mice, activation of patchy SPNs, especially dSPNs, suppressed locomotor activity, consistent with their net inhibitory role on ALDH1A1⁺ DANs and downstream motor circuits [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, the same stimulation paradigm increased locomotor speed, suggesting a functional reversal of patchy SPN output. Moreover, selective activation of patchy dSPNs alone failed to suppress movement in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, reinforcing the idea that these cells are, reduced in number, or have lost effective downstream connectivity. Meanwhile, the enhanced locomotion observed during activation of patchy iSPNs aligns with their increased proportion and GPe innervation. Together, these findings indicate that the hyperactivity observed in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice likely stems from an imbalance in patchy SPN circuitry, skewed toward indirect pathway dominance.\u003c/p\u003e\u003cp\u003eMost prior studies have attributed the locomotor effects of patchy SPNs to their modulation of DAN activity, particularly ALDH1A1\u003csup\u003e+\u003c/sup\u003e SNc DANs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In the absence of these DANs, as in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, how might reorganized patchy SPNs regulate locomotion? An early study using an independent reporter line demonstrated that patchy iSPNs preferentially target a central subregion of the GPe [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], which corresponds to a distinct subpopulation of GPe neurons associated with arkypallidal (Arky) cells, commonly marked by Npas1 or FoxP2 [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Unlike prototypical GPe neurons that engage the indirect pathway via the subthalamus nuclei, Arky neurons project back to the striatum, where they provide potent inhibition to SPNs [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The observed increase in patchy iSPN projections to the GPe in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice may result in enhanced inhibition of Arky neurons, thereby reducing their inhibitory feedback to striatal SPNs, including matrix dSPNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This disinhibition of matrix dSPNs may contribute to the hyperlocomotion observed in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice, particularly under conditions of increased patchy iSPN activity. Future studies employing electrophysiological recordings and circuit-mapping approaches will be essential to delineate how patchy iSPNs modulate Arky neuron activity in the GPe of \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice.\u003c/p\u003e\u003cp\u003eAlthough PD is defined by progressive dopaminergic degeneration, compensatory remodeling of downstream circuits may occur, particularly in early or preclinical stages. The observed increase in patchy iSPN activity may reflect an adaptive mechanism aimed at sustaining motor output in the context of dopamine loss. Despite these important insights, several limitations should be noted. First, the loss of ALDH1A1⁺ SNc DAN in \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice occurs during early postnatal development. Thus, the circuit adaptations observed in this model likely reflect developmental plasticity, which may differ fundamentally from the compensatory changes that emerge in adult-onset models or in human PD. Whether similar circuit remodeling occurs in progressive PD models or in patients remains to be determined. Second, although \u003cem\u003ePitx3\u003c/em\u003e\u003csup\u003eak/ak\u003c/sup\u003e mice display hyperactivity in open-field tests, they exhibit deficits in motor skill learning [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], suggesting that compensatory changes in the striatum are insufficient to fully restore complex motor functions. Third, our analysis was limited to anatomical projections, optogenetics manipulation and behavioral output. Future studies incorporating electrophysiological and neurochemical approaches will be critical to elucidate the functional consequences of these circuit alterations in the dorsal striatum and GPe.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe identify a novel form of striatal circuit remodeling following the developmental loss of ALDH1A1⁺ SNc DANs in mice. This reorganization selectively alters the balance of patchy SPNs, resulting in a shift toward indirect pathway dominance and functional reversal in their motor output. These findings provide mechanistic insight into how early dopamine depletion can reshape basal ganglia circuitry and highlight patchy iSPNs as potential targets for therapeutic intervention in PD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePD – Parkinson’s disease\u003c/p\u003e\n\u003cp\u003eDANs – Dopaminergic neurons\u003c/p\u003e\n\u003cp\u003eSNc – Substantia nigra pars compacta\u003c/p\u003e\n\u003cp\u003eVTA – Ventral tegmental area\u003c/p\u003e\n\u003cp\u003eALDH1A1 – Aldehyde dehydrogenase 1 family member A1\u003c/p\u003e\n\u003cp\u003eSPNs – Striatal projection neurons\u003c/p\u003e\n\u003cp\u003edSPNs – Direct-pathway striatal projection neurons\u003c/p\u003e\n\u003cp\u003eiSPNs – Indirect-pathway striatal projection neurons\u003c/p\u003e\n\u003cp\u003eGPe – Globus pallidus externa\u003c/p\u003e\n\u003cp\u003eGPi – Globus pallidus interna\u003c/p\u003e\n\u003cp\u003eSNr – Substantia nigra pars reticulata\u003c/p\u003e\n\u003cp\u003eAAV – Adeno-associated virus\u003c/p\u003e\n\u003cp\u003eChR2 – Channelrhodopsin-2\u003c/p\u003e\n\u003cp\u003etdT – tdTomato fluorescent protein\u003c/p\u003e\n\u003cp\u003eTH – Tyrosine hydroxylase\u003c/p\u003e\n\u003cp\u003eCTIP2 – COUP-TF-interacting protein 2\u003c/p\u003e\n\u003cp\u003eEYFP – Enhanced yellow fluorescent protein\u003c/p\u003e\n\u003cp\u003eDrd1 – Dopamine receptor D1\u003c/p\u003e\n\u003cp\u003eDrd2 – Dopamine receptor D2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree with the content for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. Source data are provided with this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported in part by the Intramural Research Program of the National Institutes of Health (H.C., ZIAAG000928, 000959, 000945), the Rodent Behavioral Core of Intramural Research Program of National Institute of Mental Health (MH002952), the National Natural Science Foundation of China (W.D.L., 32220103006; J.J., 32271173), the Natural Science Foundation of Beijing Municipality (J.J., 7242214), and Fujian Provincial Natural Science Foundation (B.S., 2023J01301). The contributions of the NIH author(s) were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.C. conceived and designed the study, wrote the manuscript, and prepared figures with input from all authors. J.D. designed and conducted stereotactic surgeries, optogenetics, histology, behavioral experiments, data analyses, prepared figures, and contributed to writing the methods, figure legends, and manuscript. B.T.S., B.S. and S.L. performed RNAscope in situ hybridization, data analyses, histology, mouse breeding, and behavioral testing. V.M. contributed to RNAscope image analysis, mouse breeding, and histology. L.W. performed optogenetic experiments and contributed to manuscript editing. J.K. conducted image analyses. L.T. assisted with surgeries. L.S. contributed to histological analyses. L.C. performed stereotactic surgeries. J.H.D. contributed to RNA sequencing data analysis. W.D.L. provided guidance on data interpretation and manuscript editing. J.J. made the initial observation on anatomical alterations and contributed to manuscript revision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Marten P. Smidt of University of Amsterdam for providing the PITX3 antibody, NIMH rodent behavioral core for assisting in behavioral tests, and Cai lab members for their various helps and constructive feedback.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eParkinson J: \u003cstrong\u003eAn essay on the shaking palsy.\u003c/strong\u003e \u003cem\u003eLondon: Sherwood, Nelly and Jones \u003c/em\u003e1817.\u003c/li\u003e\n\u003cli\u003eSveinbjornsdottir S: \u003cstrong\u003eThe clinical symptoms of Parkinson\u0026apos;s disease.\u003c/strong\u003e \u003cem\u003eJ Neurochem \u003c/em\u003e2016, \u003cstrong\u003e139 Suppl 1:\u003c/strong\u003e318-324.\u003c/li\u003e\n\u003cli\u003eVijayakumar D, Jankovic J: \u003cstrong\u003eDrug-Induced Dyskinesia, Part 1: Treatment of Levodopa-Induced Dyskinesia.\u003c/strong\u003e \u003cem\u003eDrugs \u003c/em\u003e2016, \u003cstrong\u003e76:\u003c/strong\u003e759-777.\u003c/li\u003e\n\u003cli\u003eBastide MF, Meissner 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Mallet N, Mohamed FY, Sharott A, Dodson PD, Nakamura KC, Suri S, Avery SV, Larvin JT, Garas FN, et al: \u003cstrong\u003ePrototypic and arkypallidal neurons in the dopamine-intact external globus pallidus.\u003c/strong\u003e \u003cem\u003eJ Neurosci \u003c/em\u003e2015, \u003cstrong\u003e35:\u003c/strong\u003e6667-6688.\u003c/li\u003e\n\u003cli\u003eBeeler JA, Cao ZF, Kheirbek MA, Ding Y, Koranda J, Murakami M, Kang UJ, Zhuang X: \u003cstrong\u003eDopamine-dependent motor learning: insight into levodopa\u0026apos;s long-duration response.\u003c/strong\u003e \u003cem\u003eAnn Neurol \u003c/em\u003e2010, \u003cstrong\u003e67:\u003c/strong\u003e639-647.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mond","sideBox":"Learn more about [Molecular Neurodegeneration](http://molecularneurodegeneration.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mond/default.aspx","title":"Molecular Neurodegeneration","twitterHandle":"@MolNeuro","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ALDH1A1, Kremen1, dopaminergic neurons, Parkinson’s disease, Pitx3, striatal projection neurons, direct-pathway, indirect-pathway, optogenetics, and neuromodulation","lastPublishedDoi":"10.21203/rs.3.rs-7401124/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7401124/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMotor symptoms of Parkinson’s disease (PD) primarily result from the degeneration of nigrostriatal dopaminergic neurons (DANs), particularly the Aldehyde Dehydrogenase 1A1-positive (ALDH1A1⁺) subpopulation. \u003cem\u003ePitx3\u003c/em\u003e-deficient mice exhibit selective developmental loss of ALDH1A1⁺ DANs but paradoxically display hyperlocomotion, suggesting compensatory changes in striatal circuitry. The dorsal striatum contains four main types of spiny projection neurons (SPNs): patch (or striosome) and matrix subtypes of both direct-pathway (dSPNs) and indirect-pathway (iSPNs). Activation of patch dSPNs suppresses locomotion by inhibiting ALDH1A1⁺ DANs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe combined RNAscope \u003cem\u003ein situ\u003c/em\u003e hybridization with SPN subtype-specific reporter mice to quantify patch and total dSPNs and iSPNs in \u003cem\u003ePitx3\u003c/em\u003e-deficient and control mice. Three patch SPN reporter lines (\u003cem\u003eKremen1\u003c/em\u003e\u003csup\u003e2A-Cre\u003c/sup\u003e, \u003cem\u003eNr4a1\u003c/em\u003e-\u003cem\u003eGFP\u003c/em\u003e, and\u003cem\u003e Pdyn\u003c/em\u003e\u003csup\u003eIRES-Cre\u003c/sup\u003e) were used to map projections. Optogenetic stimulation was performed in freely moving mice to assess the behavioral effects of activating patch dSPNs and iSPNs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePitx3-\u003c/em\u003edeficient mice showed no change in the overall dSPN:iSPN ratio but exhibited a marked shift in the patch dSPN:patchy iSPN ratio, which decreased from 1.7 in control mice to 0.7 in the \u003cem\u003ePitx3-\u003c/em\u003edeficient group. Accordingly, patch dSPN projections to the \u003cem\u003esubstantia nigra pars reticulata\u003c/em\u003e (SNr) were reduced, whereas patch iSPN projections to the \u003cem\u003eglobus pallidus externus\u003c/em\u003e (GPe) were enhanced. Notably, while optogenetic stimulation of patch dSPNs and iSPNs suppressed locomotion in control mice, the same stimulation promoted locomotion in \u003cem\u003ePitx3\u003c/em\u003e-deficient mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings reveal a selective reorganization of patch SPNs in response to developmental loss of ALDH1A1⁺ DANs, characterized by reduced patch dSPN and enhanced patch iSPN influence. This shift may underlie the paradoxical hyperlocomotion observed in \u003cem\u003ePitx3\u003c/em\u003e-deficient mice and provides insight into circuit-level adaptations with potential therapeutic relevance for PD.\u003c/p\u003e","manuscriptTitle":"Developmental Dopamine Loss Rewires Striatal Circuits to Promote Locomotion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 17:56:27","doi":"10.21203/rs.3.rs-7401124/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T23:46:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T20:49:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T16:52:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T21:45:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41282338250721975281867845336220389517","date":"2025-09-11T20:02:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143953156111760725284802623137628843436","date":"2025-09-11T03:42:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84600601628510041726877462760375818665","date":"2025-09-09T02:25:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-08T18:00:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-31T04:03:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-21T11:45:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurodegeneration","date":"2025-08-18T15:10:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurodegeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mond","sideBox":"Learn more about [Molecular Neurodegeneration](http://molecularneurodegeneration.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mond/default.aspx","title":"Molecular Neurodegeneration","twitterHandle":"@MolNeuro","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"140b57de-1c45-40d8-b634-b5f3f104de34","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:00:42+00:00","versionOfRecord":{"articleIdentity":"rs-7401124","link":"https://doi.org/10.1186/s13024-025-00920-2","journal":{"identity":"molecular-neurodegeneration","isVorOnly":false,"title":"Molecular Neurodegeneration"},"publishedOn":"2026-01-03 15:57:17","publishedOnDateReadable":"January 3rd, 2026"},"versionCreatedAt":"2025-09-15 17:56:27","video":"","vorDoi":"10.1186/s13024-025-00920-2","vorDoiUrl":"https://doi.org/10.1186/s13024-025-00920-2","workflowStages":[]},"version":"v1","identity":"rs-7401124","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7401124","identity":"rs-7401124","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}