Transcriptional and anatomical diversity of medium spiny neurons in the primate striatum.

Cell Type Taxonomy in the Primate Striatum(A) MRI image of a Rhesus macaque coronal brain section (left) showing three striatal regions labeled with cyan (caudate nucleus), brown (putamen), and pink (ventral striatum). Schematic striatum (middle) marked by Cd (caudate nucleus), Pt (putamen), and VS (ventral striatum). The right axis shows dorsal (D), ventral (V), lateral (L), and medial (M) directions.(B) UMAP visualizations of the samples from two subjects (P and F).(C) UMAP visualizations of striatal nuclei colored by the three regions. The color scheme for these regions is the same as in A.(D) Feature plots of canonical neuronal and astrocyte marker gene expression in striatal nuclei.(E) UMAP visualization colored according to eight major classes in the NHP.(F) Heat map of differentially expressed genes. Color bar at the top corresponds to the major classes identified in E.(G) Violin plots of distributions of marker gene expression across nine clusters, with MSNs divided into D1- and D2-MSNs.See also Figures S1 and S2, Table S1, and Data S1.

Medium Spiny Neuron (MSN) Subtypes(A) UMAP projection of MSN nuclei. Each dot represents a nucleus, and the colors represent the different MSN types.(B) Feature plots for the expression of DRD1, TAC1, DRD2, PENK showing the separation of D1- and D2-MSNs and the expression of marker genes enriched in each cluster.(C) Heatmap showing the top ten most enriched genes in each MSN type. Colored bar at the top corresponds to the colors in A.(D) (top) MSN type identifications colored according to A. (bottom) Violin plots showing cell type and compartment specific marker gene expression.(E) The accuracy rate between SCCAF decoded cell type and actual cell type using the data combined from both subjects.See also Figures S1 and S3, Table S1, and Data S1.

Archetypal Analysis of MSN Subtypes(A) Representative genes showing significant discontinuity between a subtype pair.(B) Representative genes showing non-significant discontinuity between a subtype pair.(C) The p-value distributions between each subtype pair.(D) Heat map showing the cosine similarity between cells within and between the nine types of MSNs.(E) FISH labeling of CRYM (magenta) and CNR1 (green) reveals a continuous gradient on the dorsal-ventral axis.(F) CRYM (top) and CNR1 (bottom) expressions along the D1/D2-hybrid archetype axes.(G) The archetype distribution of subtype pairs (top) that were divided between the dorsal and ventral striatum and CNR1 (bottom) distributions in these archetype axes.(H) Heat map showing the cosine similarity between cells within and between striosome and matrix in caudate and putamen. The color scale is the same as in D.(I) Distribution of caudate and putamen in archetype axes.(J) OPRM1 expression in the caudate and putamen cells along the archetype axes.See also Figure S3, Table S1 and S2, and Data S2.

MSN Subtypes in the Dorsal Striatum(A) FISH labeling of DRD1 (green) and DRD2 (magenta). White box indicates the region shown in B. The top right axis shows dorsal (D), ventral (V), lateral (L), and medial (M) directions.(B) High resolution image of region highlighted in A.(C) Schematic diagrams of the three sections used for DRD1 and DRD2 quantification. The square boxes indicate the quantified regions of interest (ROIs).(D) Quantification of cell density of neurons expressing DRD1 (green), DRD2 (magenta), or both (orange) in the caudate and putamen. Error bars are SD across ROIs.(E) One example MSN expressing both DRD1 and DRD2.(F) RXFP1 labels D1/D2-hybrid MSNs in the dorsal striatum. Arrowhead points to an example D1/D2-hybrid cell.(G) Quantification of DRD1 and DRD2 grain number in D1/D2-hybrid cells and normal D1- or D2-MSNs. Unpaired t-test was used for statistical analysis and p values were indicated on the plots. Error bars represent standard deviation (SD) across 6 cells per type. NS: non-specific.(H) FISH labeling of DRD1 and RXFP1. Left two pictures are original FISH images showing the distribution of DRD1 and RXFP1. The right two pictures are CellProfiler processed images showing DRD1 expressing (red dots) or DRD1 and RXFP1 (black dots, enlarged for display purposes) co-expressing cells. Grey dots are nuclei. Abbreviations: Cd, caudate; Pt, putamen; IC, internal capsule.(I) Immunohistochemistry of KChIP1 showing robust striosome pattern.(J) FISH labeling of KCNIP1 (yellow) and STXBP6 (blue) distinguishes striosome and matrix, respectively. White square indicated the region shown in K.(K) (top) Detail of the white square in J. (bottom) As in above, for characteristic striosome and matrix markers, BACH2 and GDA. Abbreviations: Cd, caudate; Pt, putamen; IC, internal capsule.See also Figure S4, Table S1, and Data S4.

MSN Subtypes in the Ventral Striatum(A) (top) Double labeling with DRD1 (left) and ARHGAP6 (middle) of the NAs shell/OT shows that ARHGAP6 is selectively enriched in the shell/OT. (bottom) CellProfiler processed images for the above images. Lettered boxes indicate regions shown in Figure S5A and S5B.(B) (left) Quantification of grain number of ARHGAP6 in shell/OT and core. Unpaired t-test was used for statistical analysis. Error bars represent standard deviation (SD) across 32 cells per each region. (right) Quantification of grain number of GREB1L in shell/OT and core. Unpaired t-test was used for statistical analysis. Error bars represent standard deviation (SD) across 29 cells per each region.(C) Violin plot showing TAC3 levels in D1Sh-TAC3 archetype, other D1Sh cells, and TAC3 interneurons(D) TAC3 co-localizes with DRD1 in medial shell MSNs.(E) DRD2 FISH image showing the outline of the striatum. Dashed white line delineate the borders of striatum.(F) CellProfiler results showing the TAC3 distribution (red dots) in the section in E. Grey dots are nuclei.(G) CellProfiler results showing the distribution of TAC3 and DRD1 co-expressing cells (dark green dots) in the section in E.(H) CellProfiler results showing the distribution of TAC3 and DRD2 co-expressing cells (dark green dots) in the section in E.(I) CellProfiler results showing the distribution of TAC3 and DRD1 co-expressing cells in a second animal (dark green dots).See also Figure S5, Tables S1 and S3, and Data S3 and S4.

Cell Types in the Interface Islands(A) FISH stain of DRD1 (green) and DRD2 (magenta). Inset: White area indicates striatum and the red box highlights the area shown in A and B.(B) FISH stain of DRD1, RXFP1 and CPNE4 in immediately adjacent section from A.(C) High-resolution image of the regions indicated with the letter “C” in B.(D) High-resolution image of the region indicated with the letter “D” in B.(E) Distribution of RXFP1 and CPNE4 clusters across six rostral-caudal regions identified by multichannel FISH. The upper right axis shows dorsal (D), ventral (V), lateral (L), medial (M), rostral (R), and caudal (C) directions. Dashed white box denotes RXFP1 clusters in putamen, the FISH image of which is shown in Figure S5H. Yellow arrowheads point to RXFP1 clusters in caudal extent of NAc in both illustration and images shown in F.(F) Example RXFP1 clusters in caudal extent of NAc. AC = anterior commissure, GPe = external globus pallidus, VP = ventral pallidum.See also Figure S5-S7, Table S1, and Data S4.

μ-Opioid Receptor Expression is Specifically Enriched in D1-NUDAP cells(A) FISH stain of DRD1 and RXFP1 as well as OPRM1 in two close sections. White dashed line delineates the boundaries of a RXFP1 cluster.(B) Quantification of grain number of OPRM1 in RXFP1 clusters and close D1-MSNs. Unpaired t-test was used for statistical analysis. Error bars represent standard deviation (SD) across 41 cells in each group.(C) MOR expression in an adjacent section of A.(D) FISH stain of DRD1 and CPNE4 as well as OPRM1 in two close sections. White dashed line delineates the boundaries of a CPNE4 cluster.(E) Quantification of grain number of OPRM1 in CPNE4 clusters and close D1-MSNs. Unpaired t-test was used for statistical analysis. Error bars represent standard deviation (SD) across 36 cells in each group.(F) MOR expression in an adjacent section of D.See also Figure S7, Table S1, and Data S4.