Article Figures & Data
Figures
- Figure 1.
Clustering analysis reveals all major striatal cell subtypes. A, Time course of experiment and graphical description of experimental groups for snRNA-seq experiments. B, ALO AIMs and (C) contralateral rotations (±SEM) for the first 60 min of the final behavioral session for each group of animals before tissue harvesting. D, Percentage of TH+ cells in nigral sections on intact and DA lesioned side (±SEM). E, Integrated UMAP of striatal cellular subclustering across all experimental groups (>97,000 nuclei). F, Dot plot examining % of nuclei expressing cluster-defining genes (size of the dots) and their average expression (color of the dots) within each cluster. See Extended Data Tables 1-1 and 1-2 for more details.
- Figure 2.
l-DOPA treatment alters major and minor subpopulation proportions and transcription within striatal cells. A, Representative UMAPs of the striatal cellular distribution within each experimental group. B, Heat map depicting the population proportions of major and minor cellular subpopulations within each l-DOPA exposure group depicted as a percentage of the population sizes of the DA-lesioned, vehicle-treated group. See Extended Data Table 2-1 for more details. C, Effect of l-DOPA exposure for 1 (Acute), 5 (Subchronic), or 10 d (Chronic) on the number of discrete differentially expressed genes (DEGs; log2 fold change > 0.10) depicting either decreased or increased transcription in each striatal cell subpopulation. See Extended Data Table 2-2 for more details.
- Figure 3.
MSN subpopulations are specifically altered by l-DOPA exposure that leads to LID. A, Representative UMAPs of the striatal MSN populations within each experimental group. B, C, Population ratios of D1-MSN (B) and D2-MSN (C) subpopulations compared with intact, l-DOPA-naive striata. D, UMAPs of expression of the immediate early genes Arc, Fos, and Fosb in each experimental group. The order of UMAPs from left to right follows that depicted in A. E, Gene module analysis of integrated MSNs utilizing MonocleTM. See Extended Data Table 3-1 for more details. F, Expression profiles for genes within Modules 3, 4, 6, and 9 that were enriched in Act MSNs. Selected module genes are listed next to UMAPs for each module in italics. Selected significant terms from gene ontology analysis for Modules 3, 4, 6, and 9 are denoted beneath each UMAP. See Extended Data Table 3-2 for more details. G, Expression profile of DEGs observed in RNA-seq derived from a Drd1a-BAC-TRAP rat model of LID (Heiman et al., 2014) mapped onto MSNs from the current snRNA-seq dataset. Left side, Violin plot depicting the ratio of coexpressed DEGs within integrated MSN subpopulations. Right side, Mapped enrichment of the MSNs from the current dataset compared with that published by Heiman et al. (2014). H, Expression profile of DEGs induced in hippocampal cells by retrieval of a previously entrained stimulus–response association observed in nRNA-seq derived from an Arc-BAC-TRAP mouse model of associative learning (Marco et al., 2020) mapped onto MSNs from the current snRNA-seq dataset. Left side, Violin plot depicting the ratio of coexpressed DEGs within integrated MSN subpopulations. Right side, Mapped enrichment of the MSNs from the current dataset compared with that published by Marco et al. (2020).
- Figure 4.
Transcriptional effects of DA lesion and LID development and stable expression revealed by the pseudobulk-seq analysis of D1-MSNs. Naïve, lesioned (green) compared with (A) acute (red; 1 d) l-DOPA-treated, lesioned or (B) repeated (blue; 5 and 10 d) l-DOPA-treated, lesioned striatal D1-MSNs. C, Acute compared with repeated l-DOPA-treated, lesioned striatal D1-MSNs. (1) Volcano plots depicting DEGs (log2 fold change > 0.10) with selected genes highlighted. (2) Selected significantly enriched terms chosen from GO analyses of DEGs enriched in l-DOPA-treated groups. (3) LISA-generated enriched TFs associated with DEGs. See Extended Data Tables 4-1–4-4 for more details.
- Figure 5.
Inhba/Activin/ALK4 signaling is enhanced by repeated l-DOPA treatment in Fos+ D1-MSNs and modulates LID development. A, UMAPs of the Inhba and Tgfb1 mRNA expressions in each experimental group. B, Confocal dorsal striatal images (20×) depicting enhancement of the Inhba transcript in Fos+/Drd1+ cells in animals treated for 10 d l-DOPA compared with those treated for 0 or 1 d with l-DOPA. C, QuPath-assisted quantification of confocal images depicting the Inhba, Fos, and Drd1 transcript coexpression in hemiparkinsonian mice treated for 0, 1, or 10 d l-DOPA. D, Time course of the experiment and graphical description of experimental groups utilized to establish the functional role of ALK4/TGFb1R signaling in LID development. E, Effects of ALK4 inhibition with SB431542 on summed ALO AIMs (top) and contralateral rotations (bottom) observed on Days 1, 3, 5, and 10 of cotreatment with l-DOPA. F, Confocal images (20×) and insets with arrows depicting pSMAD4 localization in NeuN+ and Iba1+ cells in the dorsal striatum of hemiparkinsonian mice treated with SB421542 (0 or 4.2 mg/kg) and l-DOPA for 10 d and QuPath-assisted quantification of confocal images depicting the pSMAD4, NeuN, and Iba1 coexpression in mice treated with SB421542 (0 or 4.2 mg/kg) and l-DOPA for 10 d.
Extended Data
Table 1-1
Marker genes utilized for identification of cellular subtypes. Download Table 1-1, XLSX file.
Table 1-2
Cluster defining genes in order of population dominance. Download Table 1-2, XLSX file.
Table 2-1
Effect of 6-OHDA lesion and exposure to L-DOPA treatment on discrete populations of striatal cell subtypes in order of population prevalence. Download Table 2-1, XLSX file.
Table 2-2
Differentially expressed genes (adjusted p value <0.05, absolute log fold2 change >0.10) by group versus vehicle-treated, dopamine-lesioned striata. DEGs with increased expression are in red, decreased expression in blue. Download Table 2-2, XLSX file.
Table 2-3
Download Table 2-3, XLSX file.
Table 3-1
Module analysis of MSNs by MonocleTM. Download Table 3-1, XLSX file.
Table 3-2
Gene Ontology and transcription factor enrichment analyses of modules 3, 4, 6, and 9 from Extended Data Table 3-1. GO was conducted using Metascape. Transcription factor analyses conducted via LISA CistromeDB ChIP-seq database on top 200 genes (based on LogFC value). Download Table 3-2, XLSX file.
Table 4-1
Pseudobulk-seq of D1-MSNs by group. Download Table 4-1, XLSX file.
Table 4-2
Gene Ontology analyses of D1-MSN pseudobulk-seq from Extended Data Table 4-1. GO was conducted using Metascape. Download Table 4-2, XLSX file.
Table 4-3
Transcription factor enrichment of DEGs from Extended Data Table 4-1 conducted using LISA CistromeDB ChIP-seq database on top 200 genes (based on LogFC value). Download Table 4-3, XLSX file.
Table 4-4
Differentially expressed genes by subcluster in DA-lesioned, Acute (1 d L-DOPA) versus DA-lesioned, Chronic (10 d L-DOPA) striata. DEGs with increased expression are in red, decreased expression in blue. Download Table 4-4, XLSX file.
