PROJECT SUMMARY/ABSTRACT Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic disorder characterized clinically by recurring episodes of pelvic pain and increased urination frequency, significantly impairing patients' quality of life. Irritable bowel syndrome (IBS) is a multifactorial disorder characterized by abdominal pain and altered bowel habits, as well as other somatic, visceral, and psychiatric comorbidities. Despite distinct origins, recent studies have shown a strong overlap between both syndromes and this pathological co-occurrence is believed to be responsible for the comorbidity of a number of lower urinary tract and colonic disorders, including, but not limited to, IBS, inflammatory bowel disease (IBD), overactive bladder (OAB), and IC/BPS. There is an unmet medical need in terms of effective diagnostics and treatment. One of the challenges is a lack of understanding of the molecular and cellular mechanisms underlying the pathogenesis of neurogenic bladder pain. Moreover, how bladder inflammation promotes colon sensitization is poorly understood. Pilot studies showed that MrgprB2 is required for bladder pain and sensitization of colon to mechanical stimulation induced by intravesical instillation of LL-37. These observations led us to frame the hypothesis that MrgprB2+ mast cell (MC)-TRPV1+ nociceptor clusters mediate bladder pain and inter-organ sensitization between the bladder and colon. In this grant proposal we will: 1) Use chemogenetic inhibition of mast cells and virally mediated ablation of TRPV1+ bladder-innervating nociceptors to investigate in vivo functions of the MrgprB2+ MC-TRPV1+ nociceptor clusters in MrgprB2-dependent bladder pain; use humanized mouse model with targeted expression of MrgprX2 in MCs to test if human MrgprX2 also impacts bladder pain; use TRPV1+ nociceptor specific Piezo2 cKO mice and virally mediated ablation of Piezo2 function in bladder-innervating nociceptors to determine if nociceptor-expressed mechanosensitive Piezo2 channels mediate bladder pain induced by various irritants including LL-37, compound 48/80, or CYP. 2) Focus on differentiating visceral nociceptors innervating both the bladder and colon and use unique chemical- and viral-mediated approaches to either ablate or chemogenetically inhibit these distinct nociceptors to determine if they are required for the generation of colon pain induced by bladder inflammation caused by LL-37, compound 48/80, or CYP; test if MC-derived LTC4-neuronal CysLTR2 axis contributes to bladder pain and mediates inter-organ cross sensitization in the colon. Our findings will provide cellular and molecular basis for bladder pain and bladder-colon cross sensitization mediated by the MrgprB2+ mast cell-TRPV1+ nociceptor clusters and will undoubtedly lead to new therapeutic approaches for treating visceral pain.