Project Summary: Opioid dependence is a chronic and relapsing disease and characterized by compulsive drug seeking and use. Opioid dependence results in excessive degrees of physical dependence (withdrawal syndrome). Although psychological dependence on opioids is an important feature of dependence/abuse, physical dependence itself (withdrawal syndrome) is a major cause of compulsive drug-taking behavior and short term relapse. Unfortunately, the mechanism of opioid in the pathological changes in opioid dependence is not yet understood in detail. Recent studies show that chronic morphine induces release of proinflammatory cytokines (e.g. TNF). Oxidative stress may participate in the process of dependence /withdrawal of heroin. However, very few studies have addressed the precise mechanisms by which these glial elements contribute to CNS neuronal TNFR, calcium and ROS response in opioid withdrawal. The role of the PAG in the expression of opioid withdrawal has been suggested by studies, but we know little of the detailed molecular mechanisms. Here, we hypothesize that glial activation resulting in the release of proinflammatory cytokines, and that neurons activated by cytokine receptor induce mitochondrial ROS and pCREB (a key protein in drug abuse behavior) in the development of morphine withdrawal. Specific Aim 1, To test whether glial TLR4 activation induces release of cytokine in morphine withdrawal. Specific Aim 2, To test whether activation of cytokine receptor induces mitochondrial ROS in morphine withdrawal in the PAG. Specific Aim 3, To test whether mitochondrial ROS in the PAG is involved in MW through the pCREB. The proposal will use assays of pharmacology, molecular biology, immunohistochemistry, cell type-targeting gene transfection, and imaging density analysis to complete the novel hypothesis. The results of these studies will provide important insights into the pathogenesis of opioid abuse, and may provide preclinical evidence for a novel therapy of opioid dependence.