In order to map the distribution of D1-RXFP1 and D1- CPNE4 islands, we triple labeled six sections spaced at 750 μm intervals with probes against DRD1, RXFP1, and CPNE4 in monkey B. We scanned the whole sections and ran a custom CellProfiler pipeline87 to determine the spatial distribution of nuclei and signal intensity of individual channel for each nucleus. We marked regions as D1-RXFP1 (D1-CPNE4) that had a majority of cells double-labeled with DRD1 and RXFP1 (CPNE4). To confirm that these islands were D1 exclusive, we double-labeled sections with DRD1 and DRD2 adjacent to the above six sections. We confirmed that these islands are exclusively the D1 clusters from the similar CellProfiler analysis. We did the similar process for eight sections spaced at 600 μm intervals for monkey K.
