Four MSN clusters were highly enriched in the nuclei from the VS (Figure 2D). Three of the clusters were DRD1 positive, whereas the fourth cluster was DRD2 positive. Differential gene analysis of the two largest clusters (Figure 2A, light and dark green) revealed selectively enriched marker genes, including GREB1L, ARHGAP6, and GRIA4 (Figure 2C). FISH labeling of the striatum with DRD1 and ARHGAP6 revealed that ARHGAP6 was enriched in a restricted portion of the VS that likely corresponds to the NAc shell and OT (Figures 5A, 5B and S5A), but was not prevalent in the core (Figure S5B). Quantification of grain number of ARHGAP6 in putative shell/OT and core regions confirmed that ARHGAP6 was more enriched in shell/OT (Figure 5B, left), and very similar pattern was observed with probes against another enriched marker gene, GREB1L (Figure 5B, right). We labeled sections with DRD1 and GREB1L probes and an adjacent section with an immunofluorescent stain for calbindin, which roughly markes the border between NAc core and shell.33 The GREB1L intensity traced the putative transition from the calbindin-poor shell to the calbinin-rich core (Figure S5C). Thus, these results indicate that NAc Shell and OT are comprised of region-specific D1- and D2-MSNs.
