Cell type-specific pharmacology of NMDA receptors using masked MK801
- Janelia Research Campus, Howard Hughes Medical Institute, United States
Figures
Figure 1
Cell type specific pharmacology for NMDA-R inhibition.
(A, B) Strategy for cell type-specific targeting of a masked MK801 molecule to a defined subpopulation of neurons in brain tissue that transgenically express an unmasking enzyme. The masked MK801 enters every cell but MK801 is unmasked in only those cells that transgenically express the enzyme porcine liver esterase (PLE). (B) The intracellularly liberated MK801 can block NMDA-Rs in the PLE-expressing neurons. Yellow triangle, glutamate. (C) Synthesis of CM-MK801. (a) chloromethylchlorosulfate; (b) NaI; (c) 4-hydroxy-3-nitrobenzaldehyde, 44% for a-c; (d) NaBH4, 48%; (e) 4-nitrophenyl chloroformate, 65%; (f) MK801, 32%. (D) Enzymatic hydrolysis of CM-MK801 by PLE is followed by spontaneous 1,6-elimination to liberate MK801.
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.
Figure 3
Cell type-selective blockade of cocaine-induced synaptic plasticity in dopamine neurons.
(A) Schematic diagram of cocaine-induced synaptic plasticity. Cocaine increases extracellular dopamine which leads to NMDA-R dependent upregulation of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. It is unclear if this is through NMDA receptors on DA neurons or through NMDA-R on other cell types that may have a subsequent effect on AMPA-R activity in DA neurons. (B) Confocal images of ventral tegmental area (VTA) brain slices from dopamine transporter Cre (Slc6a3Cre) mice transduced with Cre-dependent viruses expressing either PLE and mCherry (left) or EGFP (right) on opposite sides of the VTA. Scale, 50 μm. (C) Experimental protocol where VTA-containing brain slices were incubated with CM-MK801 for 40 min, followed by addition of cocaine for 10 min and washout. 3–5 hr later, miniature excitatory postsynaptic AMPA-R currents (mEPSCs) in DA neurons expressing either GFP (PLE−) or mCherry (PLE+) were recorded. Inset, images of (left) patch pipette recording from dopamine neuron (right) expressing mCherry and PLE. Scale, 5 μm. (D) For brain slices treated with CM-MK801, AMPAR-mediated mEPSC amplitude was increased in PLE− VTA DA neurons following cocaine treatment. Synaptic potentiation was blocked in PLE+ neurons treated with cocaine, and the mEPSC amplitude was similar to that in PLE+ or PLE− neurons that were not exposed to cocaine. (E) mEPSC frequency was unaffected by cocaine or CM-MK801. (F) Resting neuronal membrane potential (Vm), input resistance (Rin), and membrane capacitance (Cm) were not changed by expression of PLE, or exposure to cocaine and CM-MK801. Data is represented as mean and error bars indicate s.e.m.
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Cell type-specific pharmacology of NMDA receptors using masked MK801
eLife 4:e10206.
https://doi.org/10.7554/eLife.10206
