The project studies ketamine, its metabolites, and related drugs [Ketamine-Class Antidepressant Drugs (KCADs), our term]. Sub-anesthetic doses of ketamine produce antidepressant effects in just a few hours (2 h) via unknown mechanism(s). However, higher doses have adverse effects. Understanding the mechanism mediating KCAD antidepressant activity is an important step in the process of drug development. This research program has the goal to fundamentally change our understanding of how this rapid antidepressant mechanism works and holds promise for development of more robust and safer treatments. The molecular target(s) of KCAD action are not known. In the absence of such knowledge, one should investigate possible actions in the compartments where KCADs are most concentrated. We test the hypothesis that the effects of KCADs in the brain are mediated, in least in part, by the accumulation of KCADs in various subcellular compartments (organelles), including synaptic vesicles. Aim 1 develops a family of next-generation genetically encoded “Intensity-based Ketamine-Sensing Fluorescent Reporters” (iKetSnFRs) for KCADs. These will dynamically image and quantify the presence of KCADs at sub-cellular levels. Aim 1 measures the time course of KCAD entry and exit from various organelles after the drugs appear or disappear near cells. Aim 2 tests the hypothesis that the antidepressant mechanism of KCADs involves accumulation in the lumen of acidic vesicles, especially in synaptic vesicles, followed by synaptic stimulation-induced release of KCAD from presynaptic terminals. Aim 3 detects KCAD-induced neurotransmitter release from presynaptic terminals, employing next generation genetically encoded biosensors for various neurotransmitters. The experiments also include electrophysiological studies of membrane and synaptic properties. The data from Aims 1, 2 and 3 will not in themselves develop a new ~2 h antidepressant drug. But the data from the proposed experiments can help to understand how KCADs exert their effects. The data will also guide the development of related molecules with fewer potential side effects as new fast-acting therapies for depression.