(A-D) Confocal images from representative DRN coronal sections stained for transcripts of interest using multiplexed single molecule FISH. All sections were stained for Slc6a4 (blue) to identify 5-HT neurons, and counterstained using DAPI (gray). For panels A and B, regions outlined in yellow are enlarged in the images on the right labeled (i), (ii), and (iii), which correspond to the ventromedial DRN (vmDRN), dorsomedial DRN (dmDRN), and dorsolateral DRN (dlDRN), respectively. (A) 5-HT neurons expressing Slc17a8 (green) are enriched along the midline in both vmDRN and dmDRN. 5-HT neurons expressing Gad1 (magenta) were found in dmDRN and dlDRN. (B) Cbln2 (green) is expressed in 5-HT neurons along the midline, but is largely absent from dlDRN. 5-HT neurons expressing both Cbln2 and Pdyn were also found in both vmDRN and dmDRN. 5-HT neurons in dmDRN expressed higher levels of Pdyn compared to Pdyn+ 5-HT neurons in vmDRN. (C) Putative 5-HT-V neurons expressing high levels of both Cbln2 (green) and Trpc3 (magenta) were found in the posterior B6 subregion of the DRN, which is enlarged in the image on the right. Cells co-expressing Cbln2 and Trpc3 also showed reduced staining for Slc6a4 transcripts compared to 5-HT neurons in other subregions, consistent with reduced expression of Slc6a4 in the 5-HT-V cluster from the scRNA-seq data. (D) Putative 5-HT-I neurons expressing Trh (green) are a subset of Prkcq+ (magenta) 5-HT neurons. Prkcq+;Trh- 5-HT neurons are putative 5-HT-II neurons. (E) Spatial distribution of 5-HT neurons from a single FISH experiment co-staining for Cbln2, Pdyn, and Slc6a4 (n = 5,806 cells, 3 mice). The locations of 5-HT neurons positive for Cbln2 only (blue; n = 1,926 cells), Pdyn only (green; n = 1,048 cells), or double-positive for both transcripts (magenta; n = 1,517 cells) are displayed as probability heatmaps overlaid on the coronal averaged Nissl template. (F) Probability heatmaps from a separate FISH experiment co-staining for Trh, Pdyn, and Slc6a4 (n = 6304 cells, 3 mice). Heatmaps show the spatial distribution of 5-HT neurons positive for Trh only (red; n = 355 cells), Pdyn only (green; n = 1,867 cells), or double-positive for both transcripts (orange; n = 339 cells). Scale bars: 500 µm, unless otherwise stated.