Pain can be classified into physiological and pathological (clinical) pain. Physiological pain refers to the perception after potential harmful stimuli, while pathological pain is caused by tissue damage/inflammation (inflammatory pain), or nerve lesion (neuropathic pain). Pathological pain is characterized by two forms of pain hypersensitivity: a reduced pain threshold (allodynia), and greater pain responses (hyperalgesia). Despite its short-term benefits to tissue healing, pain hypersensitivity may persist long after healing, leading to debilitating chronic pain. Pathological and chronic pain affects over 100 million Americans and costs $500-650 billion each year in health care and lost productivity. One major cause of pathological and chronic pain is nociceptor sensitization, a process that is poorly understood. Our over-arching hypothesis is that nociception is an ancient property of the nervous system, and that studying nociceptor sensitization in Drosophila will rapidly uncover conserved molecular machinery that modulates nociceptor excitability in response to tissue injury. We have pioneered the use of Drosophila to study nociception and nociceptor sensitization in vivo. In preliminary work we have shown that Drosophila nociceptors exhibit robust nociceptor sensitization after tissue injury, that this is mediated by conserved substance P and transient receptor potential (TRP) channel-based mechanisms, and that we can explore mechanistic basis of nociceptor sensitization with single-cell resolution in vivo. Our long-term goals are to define a complete cellular and molecular picture of how tissue injury potentiates nociceptor responses to pain stimuli. Applying combinatorial tools including molecular genetics, cell biology, biochemistry, electrophysiology and imaging, we propose in the current application: 1) the cellular and molecular characterization of nociceptor sensitization; 2) to elucidate mechanisms by which regulation of dTRPA1 (Drosophila TRPA1) splice isoforms causes nociceptor sensitization; 3) to determine if dTRPA1 splice isoform regulation is the target of inflammatory molecules (referred to as pain sensitizers). Our proposed studies will test the exciting hypotheses that pain sensitizers released from injured tissues enhance the nociceptor sensitivity towards sensory stimuli by regulation of functionally interacting dTRPA1 isoforms. We expect that results from exploring our novel hypotheses will have important contribution to our understanding of the biology of nociceptor sensitization in Drosophila and mammals including human, and provide candidate drug targets to alleviate inflammatory and chronic pain conditions.