The goal of this proposal is to define the role of each component of the ARTN/GFR3/TRPM8 signaling pathway in radiation-associated pain (RAP). A common consequence of radiotherapy for head and neck cancer (HNC) is acute orofacial RAP. This pain is often severe, and difficult to control with current analgesics. Control of RAP relies on opioids in >80% of these cases. These drugs are not fully effective in most patients, and 6 months after finishing radiotherapy, one-third of HNC patients will still be opioid-dependent. There is an urgent need to identify safer, more effective, and less addictive pain relief strategies for HNC patients undergoing therapeutic irradiation. Addressing this need is prevented by a critical gap in our knowledge: the molecular and cellular mechanisms that drive acute orofacial RAP are unknown. In a mouse model of RAP, our preliminary data have indicated that a critical component of acute orofacial RAP is a signaling pathway which is usually identified as a “cold pain” pathway. The hypothesis to be tested in this proposal is that locally irradiated tissues generate extreme pain via a neural circuit that involves the “cold pain” pathway. This signaling pathway is principally mediated by neurons which express a specific ion channel called TRPM8. A typical mechanism for TRPM8 activation involves release of artemin (ARTN) from injured tissues; ARTN binds its neuronal receptor, called GFRα3, which in turn activates TRPM8. The objectives of this grant are: (1) to define the role of TRPM8 in mediating acute orofacial RAP; and (2) to determine whether ARTN/GFRα3 signaling is required for RAP signaling. We will test the hypothesis and achieve our objectives with three specific aims (SA): (SA1) to determine whether TRPM8-expressing neurons are critical to development of acute orofacial RAP; (SA2) to determine whether the GFRα3 receptor activates TRPM8 signaling in RAP; and (SA3) to determine whether ARTN is a critical activator of TRPM8 (and acute RAP) after oral irradiation. This contribution is significant because it will advance our understanding of the molecular and cellular mechanisms that drive acute orofacial RAP. The results will lay the groundwork for identification of new targeted treatments that will both improve comfort and reduce opioid-dependency for HNC patients with RAP. Importantly, if this RAP pathway is conserved between various anatomic sites, our results may extend well beyond the realm of HNC, and positively impact the analgesic options for patients who undergo radiotherapy for a wide range of cancers. The proposed research is innovative: there has been no research on the molecular and cellular mechanisms of RAP, and rather than attempting to alleviate pain by mitigating inflammation, we will identify strategies for direct inhibition of RAP. Our approach uses sophisticated methods for tracing neural circuits, and translational relevance is maximized through use of in vivo mouse studies, in vitro human cell culture experiments, and examination of patient-derived samples. Regardless of whether each component of our proposed mechanism fits together into a signaling cascade exactly as proposed, our work will generate novel, relevant information that advances the field toward novel therapies for acute orofacial RAP.