Ketamine persists within neuronal compartments long after systemic clearance

Ketamine produces rapid and sustained antidepressant effects after a single dose. Plasma membrane NMDA receptors are the presumptive primary molecular targets, yet how ketamine distributes, localizes, and engages neurons across cellular and subcellular compartments remains largely unknown. Ketamine, a lipophilic weak base, is thought to accumulate within acidic intracellular compartments, suggesting potential unrecognized sites of action. We developed a toolbox of genetically encoded intensity-based ketamine-sensing fluorescent reporters (iKetSnFRs) that specifically and robustly report R- or S-ketamine with cellular and subcellular resolution in vitro and in vivo, with excellent sensitivity and fluorescence change. iKetSnFR enables rapid detection of ketamine within seconds in the mouse prefrontal and visual cortex after intranasal or intraperitoneal dosing. We subsequently engineered compartment-targeted iKetSnFR variants, and in vivo imaging revealed that ketamine remains intracellularly and subcellularly engaged in neurons well after it is no longer detectable in circulation by LC-MS, at the plasma membrane of cortical neurons by iKetSnFR, or by electroencephalography. Furthermore, iKetSnFRs targeted across nine compartments reveal organelle-specific engagement of ketamine. This persistent intracellular engagement coincides with the prolonged timeframe of behavioral effects. Together, these findings link a single ketamine exposure to persistent intracellular drug dynamics and begin to provide a mechanistic basis for its rapid and enduring cellular and behavioral effects.