Project Summary: The endogenous opioid system is involved in many diverse biological processes and contributes to a range of disorders that have enormous health and societal consequences such as opioid use disorder, pain, and major depressive disorder. The endogenous opioid system consists of dozens of opioid peptides derived from three distinct precursor proteins, and three cell surface receptors that are activated by these peptides as well as by opiates and synthetic drugs. Recently, ketamine has emerged as an effective treatment for both major depressive disorder and chronic pain, and may also be effective in treating opioid use disorder. While ketamine’s anesthetic actions are due to its inhibition of a subtype of glutamate receptors, the mechanisms of its other biological effects remain elusive. Studies from our laboratory and other groups have found that ketamine interacts with the endogenous opioid system, however the full scope of these interactions is not known. Moreover, a single dose of ketamine produces rapid effects within minutes/hours, as well as sustained effects that last days/weeks. The long-term effects are remarkable and unlike the anesthetic action of ketamine which correlates with plasma levels. Our central hypothesis is that ketamine interacts with the endogenous opioid system to produce both rapid and long-term effects. This hypothesis is supported by our preliminary data showing that ketamine acts as a positive allosteric modulator of opioid receptors to synergize with endogenous opioid peptides. Our central hypothesis will be tested in three Specific Aims that are highly interrelated. Aim 1A will characterize the activity of ketamine as an allosteric modulator of the endogenous opioid system using cultured cells and membrane preparations. These studies will investigate short- and long-term effects of ketamine (including racemic ketamine and the stereoisomers S- and R-ketamine) and major bioactive metabolites (norketamine and hydroxynorketamine). Using electrophysiology in brain slices, Aim 1B will measure acute effects of ketamine on opioid receptor-mediated signaling and persistent changes induced by in vivo ketamine administration. Aim 2A will test the interaction of ketamine with highly purified preparations of opioid receptors. Aim 2B will determine the cryoEM structure of the mu opioid receptor in complex with ketamine and a peptide ligand. Aim 3 will examine the effect of ketamine on the levels of endogenous opioid peptides in mouse brain. Collectively, these studies will provide a solid understanding of the interactions of ketamine with the endogenous opioid system, providing mechanisms to account for the rapid and long-term effects of this drug. Historically, understanding the molecular targets of drugs have led to insights into the pathophysiology of disease and novel therapeutics. Because little is known regarding the mechanism(s) of ketamine as an antidepressant and analgesic, and the comorbidity of these d...