Abstract Interstitial cystitis/Bladder Pain Syndrome (IC/BPS) is a serious and painful condition of unknown etiology that affects 6% of women in the United States. The major clinical symptoms of IC/BPS are pain on bladder filling and increased urinary urgency and frequency. The majority of IC/BPS patients (90%) also suffer from comorbid anxiety and/or depression, contributing to a poor quality of life. Current available treatments for IC/BPS are largely ineffective, providing only mild symptomatic relief. Given the prevalence of this debilitating disease and the lack of effective treatments, further studies are needed to better understand the underlying mechanisms that contribute IC/BPS and those that mediate the high incidence of comorbid anxiety and depression. We previously reported that IC/BPS patients show referred abdominal hyperalgesia, a clear sign of central sensitization. In the previous funding period, we used the single nucleus RNA sequencing, spatial transcriptomics and in situ hybridization and identified two populations of CeA neurons that are activated in cystitis and appear to play opposing roles in the regulation of bladder pain. Here we will apply state of the art approaches in systems neuroscience to unravel the potential role of these unique neuronal subpopulations in the CeA in the reciprocal regulation of pain, voiding dysfunction, and negative affective behaviors (IC/BPS-like conditions). What are the respective roles of the two populations in bladder pain? Are the apparent pro- and anti-nociceptive actions of the Pde1c and Cartpt populations, respectively, restricted to referred hypersensitivity, or do they also reciprocally regulate ongoing pain? Do these populations play differential roles in the regulation of voiding behavior and negative affective behaviors? What are the critical inputs to and projections from these neurons? Do these populations undergo plasticity differentially in the induction and maintenance of cystitis? How do the dynamics of this circuit change as cystitis resolves? Here we propose to answer these questions in a series of studies that test the central hypothesis that maladaptive plasticity in these unique subpopulations of CeA neurons regulates voiding dysfunction, pain sensitization, and comorbid negative affect in models of cystitis. These studies will provide new insights into the critical role of the pro- and anti-nociceptive neurons in the CeA in bladder pain and comorbid affective disorders in the context of bladder pain syndrome. If successful, these studies will point the way to identifying pharmacological approaches to restore normal circuit function around these neurons to provide relief from bladder pain, voiding dysfunction, and comorbid anxiety and depression in patients with IC/BPS.