Major depressive disorder (MDD) is one of the most prevalent and debilitating illnesses world wide, affecting 17 percent of the population and causing enormous personal and economic burden. The impact of MDD is underscored by the limitations of currently available medications, including low response rates, treatment resistant patients, and time-lag (weeks to months) for response. These data highlight the major unmet need for more efficacious and faster-acting antidepressant agents. Recent studies demonstrate that a single low dose of ketamine, a glutamate-NMDA receptor antagonist, produces rapid antidepressant actions (2 hr) that last for up to 7 days in treatment resistant patients. This rapid action, by a mechanism completely different from typical monoamine reuptake inhibitors, may represent one of the most significant findings in the field of depression over the past 2 decades. The mechanisms underlying the rapid antidepressant actions of ketamine have not been identified, and the current application addresses this issue. Preliminary studies have found that ketamine rapidly stimulates the mammalian target of rapamycin (mTOR) pathway in rat prefrontal cortex (PFC). The mTOR cascade has been implicated in the regulation of synaptic protein synthesis and activity-dependent enhancement of synaptic strength. Preliminary findings also demonstrate that ketamine rapidly increases spine number and synaptic function of PFC neurons and produces rapid antidepressant behavioral responses in rodent models. Moreover, these actions of ketamine are blocked by rapamycin, demonstrating a requirement for mTOR signaling. Based on these findings, we hypothesize that the rapid antidepressant actions of ketamine result from activation of mTOR signaling, stimulation of synaptic protein synthesis, and increased synapse/spine formation and function in the PFC. This application describes an integrated multidisciplinary approach, including molecular, biochemical, electrophysiological, morphological, and behavioral studies to test this hypothesis. Aim 1 will characterize the time course and regional localization of ketamine-stimulation of mTOR signaling, synaptic proteins, spines number, function, and behavioral response, as well as confirm the requirement for mTOR using a combination of pharmacological, viral vector, and shRNA approaches. The hypothesis that ketamine can rapidly reverse the deficits in spine number and function resulting from chronic stress exposure will also be tested. Aim 2 will determine the role of glutamate transmission and neurotrophic factor signaling, which have been implicated in activity-dependent stimulation of mTOR and induction of synaptic strength. Aim 3 will determine if novel therapeutic targets identified in Aims 1 and 2 produce rapid ketamine-like antidepressant responses or sustain the actions of a single dose of ketamine. Characterization of the signaling pathways that underlie the actions of ketamine will provide novel targets for safer, rapid-acting antidepressants and/or agents that sustain the response to ketamine.