Neuropathic pain is a common problem among Veterans that substantially impedes their efforts to rehabilitate function following injury. Current treatments are inadequate, but our recent observations show that cannabinoids hold promise for new therapeutic approaches. Systemic administration of cannabinergic drugs has limited analgesic utility due to diverse side effects, such as unwanted psychoactive changes. However, there is growing recognition of the participation of endocannabinoids (ECs), the endogenous agonists of cannabinoid receptors, in driving CNS pain regulation through descending inhibition of sensory pathways, indicating a possible avenue for therapy. The dorsal periaqueductal gray (dPAG) is a key midbrain center for EC-driven antinociception mediated by descending sensory inhibition that is coordinated with enhanced autonomic function through sympathoexcitation. Our promising preliminary data in rats show that maladaptations of EC signaling in the dPAG, including upregulation of the catabolic enzyme fatty acid amide hydrolase (FAAH) and reduced levels of the EC N-arachidonoylethanolamine (AEA), are associated with the development of chronic pain and autonomic dysfunction after nerve injury. These findings suggest that individuals who develop chronic neuropathic pain would benefit from increased EC signaling, particularly in the dPAG. We propose to test the hypothesis that dysregulation of endogenous cannabinoid signaling contributes to the transition from acute to chronic neuropathic pain. The first objective of the proposed studies is to determine the therapeutic potential of enhanced EC signaling in the treatment of chronic neuropathic pain and, critically, in the prevention of acute pain progressing to chronic pain. To achieve this, we will test whether attenuation of hyperalgesia can be achieved in vivo with FAAH inhibition (Specific Aim 1), positive allosteric modulation or a combination of both (Specific Aim 2), for effective and safe prevention or treatment of chronic neuropathic pain. These aims will evaluate the dPAG as a site of action for enhanced EC signaling, which is a vital step towards future development of targeted delivery of therapeutic molecules for increased specificity and decreased adverse consequences. The VA is committed to pursuing a better understanding of causes and potential treatments of chronic pain in women veterans as there is no doubt that female sex heavily predisposes a subject to chronic pain. There is also convincing evidence that endogenous analgesic processes are more cannabinoid dependent in females than in males. Specific Aim 3 will address the significant health concern of the gender gap in the diagnosis and treatment of chronic pain by evaluating mechanisms contributing to sex differences in the development of chronic neuropathic pain and its treatment through enhanced CB1R signaling.