SUMMARY Major depressive disorder (MDD) afflicts ~16% of the world population. Despite the availability of several classes and types of antidepressant medications, patients typically take many weeks, if not months, to respond to these drugs, and the majority never attain sustained remission of their symptoms. A remarkable development for the pharmacological treatment of MDD is the finding that the non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, ketamine, is an effective, rapidly acting antidepressant in treatment-refractory patients. During our previous funding cycle, we began exploring the role of ketamine’s metabolites in both the therapeutic and adverse effects of ketamine. We identified behavioral, synaptic, and neurochemical effects of the (2R,6R)- hydroxynorketamine (HNK) metabolite. In contrast to ketamine, (2R,6R)-HNK has low affinity for the NMDAR, which is consistent with its reduced adverse effects as measured in preclinical studies. We have also found that (2R,6R)-HNK enhances excitatory synaptic transmission in the hippocampus through a concentration- dependent, NMDAR activity-independent increase in glutamate release probability. Our long-term goal is to elucidate the biological activities of (2R,6R)-HNK, as well as ketamine’s eleven additional HNK metabolites, and utilize our findings to develop novel, effective compounds for the treatment of depression. The central hypothesis is that HNKs exert an acute, synapse-selective form of presynaptic plasticity that leads to a sustained strengthening of mood-relevant circuits. In Specific Aim #1 we will use slice electrophysiology to resolve the synaptic actions of (2R,6R)-HNK, and identify the mechanism(s) by which (2R,6R)-HNK acutely enhances the probability of synaptic glutamate release. We hypothesize that (2R,6R)-HNK acts through a presynaptic cAMP- BDNF-dependent mechanism to promote glutamate release. In Specific Aim #2 we will use in vivo fiber photometry assessments of neuronal activity to determine the synaptic effects of (2R,6R)-HNK on hippocampal circuitry, specifically the Schaffer collateral synapses in the CA1 region of the hippocampus. These experiments will determine (2R,6R)-HNK’s synaptic action in an intact circuit. Finally, in Specific Aim #3 we will define, in vitro and in vivo, the relative synaptic and behavioral potencies for all 12 HNKs produced via ketamine metabolism. These experiments will define structure-activity relationships at the level of synaptic function, which will allow us to refine the structure of the HNKs, in order to optimize their antidepressant and pharmacokinetic activity. Overall, our work thus far strongly implicates an immediate drug effect on presynaptic plasticity, which when the mechanism underlying this action is clarified, will open up new avenues for novel antidepressant drug discovery based upon this mechanism. The completion of our proposed experiments will have implications for the understanding of rapid-acting anti...