Molecular profiling of nociceptive brain neurons enables precision pharmacology against pain unpleasantness

Pain is a complex experience with sensory, affective, and cognitive dimensions. An ensemble of neurons in the amygdala encodes, and is causally necessary for, the unpleasant quality of pain. To determine the molecular identity of nociceptive amygdalar neurons, we combined activity-dependent fluorescent labeling (TRAP2) and single-cell RNA sequencing (scRNA-seq) to profile the gene expression of individual neurons active during nociception. These analyses revealed 17 types and 28 subtypes of amygdalar neurons. We next established a comprehensive neuron atlas by mapping the spatial organization of the marker genes defining these amygdalar cell types. Additionally, we integrated our scRNA-seq results with prior findings about amygdalar cell-types and their functions to guide the discovery of analgesic drugs that exert their effects by modulating amygdala circuits. Specifically, we searched for G-protein-coupled receptors (GPCRs) that were enriched on amygdalar neuron types involved in pain processing. We identified more than a dozen G-protein-coupled receptors (GPCRs) differentially expressed on amygdalar neuron types involved in pain processing, including specific serotonin, vasoactive intestinal peptide, and neurotensin receptors. We tested drugs engaging these GPCR targets using preclinical pain assays and identified multiple amygdalar GPCR agonists with analgesic properties. Based on prior knowledge, we predicted that the tested drugs modulate multiple pain-related pathways, we devised a combination strategy of three drugs that enhance analgesia specifically against pain unpleasantness both across acute pain models and in a preclinical model of orofacial chronic pain. Together, our findings establish the molecular structure of the amygdala and identify highly druggable targets to treat pain unpleasantness across pain types. Our results support the concept that precisely modulating GPCR drug targets to inhibit ensembles of neurons while activating others in functionally bidirectionally organized circuits such as the amygdala result in significant analgesic benefits. We anticipate that this combination approach may improve the treatment outcomes of chronic pain patients.