Opioid use disorder (OUD) has substantial negative consequences on veterans' mental and physical health, work performance, housing status, and social function. Opioid drug abuse has reached an epidemic level in the United States. There is an increased risk of overdose death with higher daily opioid doses for increasing analgesic effect. The Veterans Health Administration (VHA) recognizes the clinical challenges to successfully prescribing opioids safely for our veterans. Morphine withdrawal (MW) is one of determinants of opiate abuse in OUD individuals, yet its mechanisms are poorly understood, and effective therapies are still lacking. MW activates astrocytes to release neuroinflammatory factors. Emerging evidence shows that neuroinflammatory factor Lipocalin 2 (LCN2) from reactive astrocytes is associated with a variety of nervous system injuries and neuroinflammation. Our preliminary data showed that spontaneous MW (S-MW) induced TLR4, NF-kB (RelA/p65), and LCN2 from astrocytes. LCN2 receptor (LCN2-R) is located in the brainstem periaqueductal gray (PAG) neurons in S-MW. Epigenetic writer EZH2 silences gene expression by generating a methylated epigenetic mark at H3K27me3. Our preliminary data showed that S-MW increased neuronal EZH2 and pCREB, and decreased anti-oxidative mitochondrial sirtuin 3 (Sirt3) in the PAG. The exact molecular mechanisms of astrocytes to neuron activity in S-MW remain poorly understood. In the proposal, we will test the hypothesis that glial activity induces the release of astrocytes-derived LCN2, which lowers neuronal anti-oxidative Sirt3 and finally increases pCREB in the PAG in mice with S-MW. Specific Aim 1: To determine whether astrocytic activation induces the overexpression of LCN2 through TLR4 and NF-κB in the vlPAG in mice with S-MW. Specific Aim 2: To define whether LCN2-R mediates the lowered Sirt3 expression leading to pCREB increases in the vlPAG neurons in mice with S-MW. A crucial feature of our work is the ability to causally use new genetic/epigenetic and molecular assay, cell-type specific conditional knockout (cKO) mice, cell type-selective viral-mediated gene transfer, and molecular-pharmacological approach. The proposal will provide important insights into the pathogenesis of opioid withdrawal, and shed light on a novel therapeutic target for opioid withdrawal of opioid withdrawal. The study will could lead, in the future, to the development of new drugs for both the general population and veterans, based on our identification of the mechanism of action of TLR4---LCN2---Sirt3---pCREB pathway during opioid withdrawal.