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.
Psychedelic Science