PMID- 34605820 OWN - NLM STAT- MEDLINE IP - 175 TI - Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization. LA - eng PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Video-Audio Media PL - United States TA - J Vis Exp JT - Journal of visualized experiments : JoVE JID - 101313252 IS - 1940-087X (Electronic) LID - 10.3791/62945 [doi] FAU - Bob-Manuel, Johnson AU - Bob-Manuel J AD - Center for Hypothalamic Research and Department of Internal Medicine, UTSouthwestern Medical Center at Dallas. FAU - Gautron, Laurent AU - Gautron L AD - Center for Hypothalamic Research and Department of Internal Medicine, UTSouthwestern Medical Center at Dallas; Laurent.Gautron@UTSouthwestern.edu. IS - 1940-087X (Linking) RN - 0 (Carrier Proteins) RN - 0 (Nerve Tissue Proteins) RN - 0 (Prdm12 protein, mouse) RN - 0 (Receptors, Ghrelin) SB - IM MH - Animals MH - Carrier Proteins MH - In Situ Hybridization MH - Mice MH - Nerve Tissue Proteins MH - *Neurons, Afferent MH - *Nodose Ganglion MH - Receptors, Ghrelin/metabolism MH - Vagus Nerve PMC - PMC9235148 DCOM- 20211015 LR - 20220716 DP - 20210920 DEP - 20210920 AB - This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.