Ketamine persists within neuronal compartments long after systemic clearance

Technical Summary: Ketamine Persists within Neuronal Compartments Long After Systemic Clearance

Problem Statement
Ketamine is a rapid-acting antidepressant (RAAD) with sustained efficacy following a single dose, yet the mechanisms underlying its rapid onset and long-lasting behavioral effects remain poorly understood. While plasma membrane NMDA receptors are considered primary targets, the spatiotemporal distribution of ketamine within the brain and specific cellular compartments is largely unknown. Traditional pharmacokinetic (PK) studies rely on blood plasma measurements, which fail to capture drug dynamics at the neuronal or subcellular level. Furthermore, ketamine is a lipophilic weak base (pKa ~7.3), suggesting it may accumulate in acidic intracellular organelles (lysosomal trapping), potentially creating unrecognized sites of action that persist after systemic clearance. Current tools lack the spatial and temporal resolution to track ketamine entry, distribution, and retention within living neurons in real-time.

Methodology
To address these gaps, the authors developed a suite of genetically encoded, intensity-based fluorescent biosensors (iKetSnFRs) capable of detecting ketamine enantiomers with cellular and subcellular resolution.

• Biosensor Engineering: Building on the iNicSnFR scaffold (a mutated acetylcholine-binding periplasmic binding protein fused to circularly permuted GFP), the team utilized iterative site-saturated mutagenesis (SSM) to generate high-performance variants.
  • iSKetSnFR3.0: Engineered for high sensitivity and selectivity toward S-ketamine (EC50 ~175 nM, S-slope 20 µM⁻¹).
  • iRKetSnFR2.0: Engineered for R-ketamine detection (EC50 ~155 nM, S-slope 31 µM⁻¹).
  • Both sensors demonstrated nanomolar sensitivity, minimal cross-reactivity with metabolites (norketamine, hydroxynorketamine, dehydronorketamine), and robust fluorescence changes (ΔF/F₀).
• Subcellular Targeting: The sensors were modified with specific localization signals to target distinct compartments: plasma membrane (PM), cytoplasm (with nuclear exclusion signal, NES), nucleus (nuclear localization signal, NLS), endoplasmic reticulum (ER), Golgi, mitochondria, peroxisomes, autophagosomes, and F-actin.
• In Vitro Validation: Sensors were expressed in Neuro2a and SH-SY5Y cells, as well as primary cortical neurons. Kinetics were measured via widefield epifluorescence and confocal microscopy to determine uptake rates, washout kinetics, and pH dependence.
• In Vivo Imaging: Adeno-associated viruses (AAV1.hSynapsin) were used to express cytoplasmic, nuclear, and PM-targeted sensors in Layer 2/3 neurons of the mouse anterior cingulate cortex (ACC) and primary visual cortex (V1). Two-photon time-lapse imaging was performed in awake, behaving mice following intraperitoneal (i.p.) or intranasal (i.n.) administration of ketamine.
• Comparative Analyses: In vivo sensor signals were benchmarked against:
  • Liquid chromatography-mass spectrometry (LC-MS) of plasma to measure systemic clearance.
  • Electroencephalography (EEG) to measure cortical desynchronization.
  • Behavioral assays (open field, rotarod, hot plate) to assess functional effects.

Key Results

1. Rapid CNS Penetration: In vivo imaging revealed that ketamine enters the mouse cortex within 30–60 seconds of systemic administration, reaching peak neuronal concentrations within 5–10 minutes. This entry occurs across a wide dose range (0.1 to >100 mg/kg).
2. Intracellular Retention vs. Systemic Clearance: A critical kinetic dissociation was observed. While plasma LC-MS detected ketamine only for 45–60 minutes post-injection (with metabolites persisting longer), iKetSnFR signals indicated that ketamine remained detectable within neuronal compartments for over 2 hours.
3. Compartment-Specific Dynamics:
   • Clearance Hierarchy: Ketamine clearance was fastest at the plasma membrane, slower in the cytoplasm, and slowest in the nucleus. Nuclear signals persisted >120 minutes post-injection, whereas PM signals returned to baseline by ~90 minutes.
   • Organelle Trapping: In vitro studies showed that ketamine accumulates in organelles with varying kinetics. The Golgi (pH ~6.0–6.5) and autophagosomes exhibited the slowest washout kinetics, consistent with protonation and trapping of the weak base in acidic environments.
   • Enantiomer Differences: R-ketamine showed markedly prolonged retention in the nucleus and mitochondria compared to S-ketamine, suggesting distinct intracellular trafficking or binding mechanisms.
4. Behavioral Correlation: The prolonged intracellular retention of ketamine (>2 hours) aligned more closely with sustained behavioral effects (reduced locomotor activity) than with the transient EEG desynchronization (which returned to baseline within ~60 minutes) or plasma clearance.
5. Route of Administration: Intranasal administration also resulted in rapid cortical entry (within tens of seconds) but showed region-specific propagation delays (ACC vs. V1) and similar prolonged intracellular persistence compared to i.p. delivery.

Significance and Claims
The paper claims to provide the first real-time, subcellular resolution view of ketamine pharmacokinetics in the living brain. The authors propose that the "persistence" of ketamine within intracellular compartments—specifically the nucleus and acidic organelles—constitutes a previously unrecognized mechanism for its sustained antidepressant effects.

Key claims include:

• Reframing PK: Conventional plasma PK measurements substantially underestimate the duration of ketamine availability within the brain. Neuronal intracellular PK is a distinct determinant of drug action.
• Mechanistic Basis: The prolonged intracellular engagement of ketamine, particularly its retention in the nucleus, may underlie the long-lasting plasticity mechanisms required for sustained antidepressant efficacy, separate from the rapid NMDA receptor antagonism at the plasma membrane.
• Tool Utility: The iKetSnFR toolbox enables the selective detection of R- and S-ketamine enantiomers and their metabolites in specific cellular locales, offering a new framework for investigating the sites of action for RAADs and other lipophilic weak bases.

The study concludes that a single exposure to ketamine triggers persistent intracellular drug dynamics that extend well beyond systemic clearance, providing a mechanistic basis for its rapid onset and enduring behavioral effects.