To quantify nuclei size for RXFP1 and CPNE4 islands (Figure S6F), we first scanned high resolution images for DRD1, RXFP1, and CPNE4 triple labeled sections. We performed automatic quantification of the areas of nuclei using ImageJ: 1) adjust the threshold and convert images to black and white using the “Make Binary” function; 2) fill the holes using the “Fill Holes” function; 3) separate overlapping nuclei with the “Watershed” function; 4) then draw the ROI and automatically detect the areas of nuclei using the “Analyze Particles” function. To produce a more accurate quantification of the nuclei size, we chose to randomly sample dozens of cells expressing DRD1 in each island per section due to high packing density of the nuclei in ICjs (Figure S6E). We calculated the area of nuclei by the similar method except that we drew an area of interest around individual nucleus. Three sections in total were quantified and the area for each nucleus was normalized to the control D1-MSNs in each section.
