In previous years, we showed that the central amygdala (CeA) functions as a pain rheostat system that can amplify or suppress pain. We further showed that the directionality of pain modulation is dependent on the activity of two subsets of genetically distinct CeA neurons, with activity of one population increasing pain responses and activity of the second population decreasing pain-related responses. At the cellular level, we have also shown that these genetically distinct CeA neurons are electrophysiologically and morphologically distinct and contributed to building an agent-based computational model that allows dynamic simulation of nociceptive signal propagation through the CeA network. We have also increased the understanding of sex as a biological variable in pain processing by demonstrating important sex differences in a behavioral visceral responses, disease progression and bowel pathology in a model of colitis. In addition, we identified and functionally characterized a new efferent pain circuit from CeA-PKCδ neurons to a subthalamic structure called the zona incerta (ZI), specifically showing that injury-induced activation of CeA-PKCδ neurons monosynaptically inhibits ZI-GABAergic cells, subsequently leading to pain-related hypersensitivity. The main focus of our research program during FY24 was to investigate CeA afferent circuits contributing to pain processing, including the transition from acute to chronic pain. We published a study that showed that excitatory afferent inputs to the CeA from the parabrachial nucleus (PBN) is critical for injury-induced hypersensitivity but not acute somatosensation. In a separate study, conducted in collaboration with the Keller lab (UMB), we further showed that neurons in the PBN undergo divergent changes in excitability following nerve injury. In an additional study, in collaboration with the Neugebauer lab (Texas Tech), we demonstrated that the PB→CeA pathway and cell-type-specific hyperexcitability of CeA neurons are critical for the initiation but not the maintenance of chronic neuropathic pain, underscoring that the mechanisms for acute and chronic pain are distinct. Lastly, in collaboration with Drs. Eterovic (UCC-PR), Ferchmin (UCC-PR), Burton (UT Dallas), we published an additional study that shows a novel function of the 4R tobacco cembranoid as an analgesic agent that reduces peripheral inflammation in a sex-dependent manner. During FY24 we further began to optimize experiments that aim at studying mechanisms at the intersection of pain and affective states, including stress-induced analgesia, pain-related aversion, and pain-related affective comorbidities.