Figure 2. Cellular selectivity of CM-MK801/PLE ester/esterase pair.
(A, B) Schematic diagrams of the experimental procedure. Perforated patch voltage clamp recordings of NMDA-R synaptic currents were made on both (A) PLE/mCherry-expressing (PLE+, red) layer 2/3 (L2/3) cortical neurons and (B) adjacent neurons lacking PLE (PLE−, gray) while electrically stimulating presynaptic neurons in layer 4 (L4) in the presence of CM-MK801. (C, D) Treatment of the brain slices with CM-MK801 (5 µM) during L4→L2/3 synaptic stimulation showed gradual use-dependent reduction of the NMDA-R EPSC amplitude in (C) PLE+ but not in (D) PLE− neurons, indicating that the CM-MK801 was converted to MK801 selectively in PLE+ neurons without spilling over to adjacent PLE− neurons. Subsequent addition of the competitive NMDA-R antagonist, AP5, suppressed the NMDA-R EPSC in PLE− neurons but not in PLE+ neurons, which were fully blocked by CM-MK801. Electrical stimuli were delivered every 15 s. (E, F) Overlaid NMDA-R averaged excitatory postsynaptic currents (EPSCs) from (E) PLE+ and (F) PLE− neurons showing the initial response (black), responses after electrical stimulation (late, blue) and the response in the presence of AP5 (green). Electrical stimulation artifact reduced for clarity. (G) Grouped data for NMDA-R EPSC charge transfer (QNMDA) inhibition for PLE+ (n = 14) and PLE− (n = 17) neurons during CM-MK801 treatment and subsequent exposure to AP5. For comparison, dotted line shows QNMDA inhibition in PLE+ and PLE− neurons in the absence of CM-MK801, which is due to modest synaptic rundown. Data is represented as mean and error bars indicate s.e.m.