The rostral ventromedial medulla (RVM) has an important role in descending modulation, yet the molecular and functional diversity of RVM neurons involved in nociception remains incompletely understood. Here, we used a combination of multiplex fluorescent in-situ hybridization (FISH), anatomical tracing, and behavioral testing to examine the selective roles of Cre-labeled neuronal populations in the RVM. At the neurochemical level, we confirmed that GABAergic neurons in the RVM include serotonergic (Tph2) and MOR-expressing (Oprm1) populations and that Tac1-expressing neurons were predominantly glutamatergic. We found that nNOS-expressing neurons exhibited a mixed phenotype, co-expressing both GABAergic and glutamatergic markers. We used a series of Cre-driver lines (Fev ^Cre , Gad2 ^Cre , nNOS ^CreER , Tac1 ^Cre , and MOR ^Cre ) to visualize RVM projections and perform behavioral experiments with chemogenetics. RVM neurons exhibited divergent projection patterns to the spinal cord, and, interestingly, we identified fluorescent labeling in the superior colliculus in both MOR ^Cre and nNos ^CreER populations. Behaviorally, activation of Gad2 ^Cre neurons significantly increased thermal thresholds, supporting their anti-nociceptive role. Activation of Tac1 ^Cre neurons facilitated mechanical and thermal nociception, whereas MOR ^Cre activation suppressed scratching behaviors. Fev ^Cre and nNOS ^CreER manipulations did not significantly alter somatosensory responses. These findings highlight the molecular and behavioral complexity of RVM neurons and their distinct roles in sensory modulation.

This study reveals the molecular and functional heterogeneity of RVM neuronal subpopulations based on their neurochemical identities, projection patterns, and behavioral roles in sensory modulation. By leveraging Cre genetics and chemogenetics, we delineate cell-type-specific contributions of RVM neurons to sensory behaviors. These results highlight the complexity of descending modulatory circuits.