ABSTRACT Irritable Bowel Syndrome (IBS) and interstitial cystitis/painful bladder syndrome (IC/PBS) are common chronic visceral pain disorders that affect the colon and bladder respectively. Both conditions pose significant health and financial burden. Notably, IBS and IC/PBS present with considerable overlapping symptoms, whilst individual patients can suffer from both disorders concurrently. The colon and bladder cross-sensitize and share spinal innervations. However, despite increased understanding of their symptomatology and the role of pain afferents in their pathophysiology, there are no effective therapies. The few pharmacological drugs including opioids have significant adverse effects. Current barriers to the development of effective therapies for IBS and IC/PBS include: a) Inadequate structural and functional knowledge on the dichotomizing dorsal root ganglia (DRG) neurons of the distal colon and bladder. b) The lack of adequate investigation on sex differences in cross-organ sensitization, despite the prevalence of both conditions being higher in women than in men (female to male ratio: 2:1 for IBS, 5-10:1 for IC/PBS) c) Lack of knowledge of the sensory afferent innervation and cross-organ sensitization in species with higher translational relevance to humans. We propose to address these gaps through concurrent monitoring of the colon and bladder pain circuitries in two pre-clinical models and under 3 specific aims. 1): Structural mapping of colon and bladder pain afferent neurocircuits: Dichotomizing and non-dichotomizing neurons, their spatial distribution and connectivity. 2): Functional characterization of colon and bladder afferents in acute and chronic models of cross-organ sensitization and 3): Map spinal sites of colon and bladder pain and cross-organ sensitization through neuromodulation. We will use acute and chronic cross-organ sensitization models, state-of-the-art high- resolution imaging, 3D mapping, dual retrograde tracing of bladder and colon sensory neurons, CLARITY, ex vivo DRG Ca2+ imaging of dichotomizing neurons, ex vivo electrophysiological recordings from bladder and colon afferents and transcutaneous spinal stimulation (TSCS) approaches. The murine model will unravel the classes of pain afferents/neurons and their functional circuits in bowel-to-bladder and bladder-to-bowel acute and chronic pain cross sensitization. The porcine studies will provide novel information on the structural map of the colon and bladder dichotomizing neurons and their connections as well as cross sensitization of pain responses. It will also allow us to gain insight on the use of TSCS to map spinal cord circuits and backtrack colon and bladder pain circuits and its translational feasibility for human use. The combined multidisciplinary approaches will fill the gaps in current knowledge on the colon/bladder dichotomizing DRG neurons, spatial distribution and connections as well as the plasticity in the pain circuitries post sensitization. Our findings will contribute to the foundation for understanding colon and bladder neurocircuitries that will be critical for urgently needed drugs and/or neuromodulator therapies for chronic visceral pain syndromes such as IBS and IC/PBS.

RELEVANCE TO PUBLIC HEALTH Chronic visceral pain related to irritable bowel syndrome (IBS) and interstitial cystitis-bladder pain syndrome (IC- BPS) is one of the most common type of pain affecting over 25% of the adult US population, with a devastating effect on patients' quality of life and direct costs averaging $1.6 and $10.5 billion respectively. The proposed studies will map the peripheral structural and functional circuitries of colon and bladder pain cross-organ sensitization through tracing, function assessment and spinal neuromodulation. The outcome will fill gaps in knowledge in the neurocircuitries of pelvic pain sensitization and provide bases for new therapies and modalities, including neuromodulation, against pain in IBS and IC-BPS patients.