ABSTRACT AND PROJECT SUMMARY Many chronic pain conditions that affect craniofacial regions, such as temporomandibular disorders (TMD), disproportionately affect females. A growing number of studies show strong evidence that sex-related differences in conditioned pain modulation (CPM), a psychophysical index of endogenous pain inhibition, is one mechanism that predisposes women to an increased risk of chronic pain conditions. However, the central mechanisms underlying gender differences in CPM, as well as causal links between dysfunctional CPM and chronic orofacial pain conditions, are largely unknown. Our prior work has shown that there are sex differences in descending noxious inhibitory control (DNIC), a measure that is similar to CPM in preclinical settings, and that DNIC is modulated in a testosterone (TS)-dependent manner. The efficiency of DNIC was stronger in males compared to females. A pharmacological blockade of androgen receptors attenuated DNIC in males, and TS replacement enhanced DNIC in female rats. We also provided compelling evidence that the efficient DNIC in males is associated with a stronger resting functional connectivity between the rostral anterior cingulate cortex (rACC) and the periaqueductal gray (PAG). These observations provide a strong rationale for investigating the impact of TS on central pain modulation, and they also have significant clinical implications for pain management for both transgender and cisgender individuals undergoing hormone therapy. In this project, we will investigate the role of TS in maintaining efficient DNIC, as well as the mechanistic links between dysfunctional DNIC, TMD-like pain, and TMD-related comorbid pain conditions. Specifically, we hypothesize that the rACC to PAG circuit mediates sex differences in DNIC efficiency in a TS-dependent manner and that strengthening DNIC effectively attenuates TMD-related primary and comorbid pain responses. In specific aim (SA) 1, we will determine the role of the rACC to PAG circuit in DNIC efficiency using a behavioral paradigm and chemogenetics, which will experimentally manipulate the strength of the circuit with and without anti-androgen treatment in males and with and without TS treatment in females. In SA2, we will investigate the relationship between DNIC and TMD-like pain responses. We will conduct a concurrent functional magnetic resonance imaging (fMRI) to assess pain- induced changes in brain networks and confirm that both TS treatment and strengthening the rACC to PAG circuit rectify the pain-induced changes in the brain networks in male and female rats. In SA3, we will determine whether chemogenetically activating DNIC leads to a reduction in TMD-related comorbid pain responses and whether TS treatment further enhances the chemogenetic effects. The project will significantly improve our knowledge of the impact of sex on CNS pain modulation, which should have broad translational implications for the development of customized therapeutic st...