Vicini, Stefano, Jian Feng Wang, Jin Hong Li, Wei Jian Zhu, Yue Hua Wang, Jian Hong Luo, Barry B. Wolfe, and Dennis R. Grayson. Functional and pharmacological differences between recombinant N-methyl-d-aspartate receptors. J. Neurophysiol. 79: 555–566, 1998. N-methyl-d-aspartic acid (NMDA) receptors transiently transfected into mammalian HEK-293 cells were characterized with subunit-specific antibodies and electrophysiological recordings. Deactivation time course recorded in response to fastl-glutamate pulses were studied in isolated and lifted cells, as well as in outside-out membrane patches excised from cells expressing recombinant NR1 subunits in combination with the NR2A, NR2B, NR2C, or NR2D NMDA receptor subunits. Transfected cells were preidentified by the fluorescence emitted from the coexpressed Aequorea victoria jellyfish Green Lantern protein. Currents generated by NR1/NR2A channels displayed double exponential deactivation time course being faster than that in NR1/NR2B or NR1/NR2C channels. However, a large decay variability was observed within each cotransfection, suggesting that mechanisms additional to subunit composition may also regulate deactivation time course. NR1/NR2D channels displayed slowly deactivating currents. Channel deactivation was fast and comparable among receptors obtained by cotransfecting five distinct spliced variants of the NR1 subunit, each with the NR2A subunit. Additionally, recovery from desensitization was slower for NR1/NR2B than for NR1/NR2A channels. The average deactivation time course of responses to brief l-glutamate applications in cells where NR1/NR2A/NR2B cDNAs were cotransfected at variable ratio was intermediate between those of the NR1/NR2A and NR1/NR2B channels. Although immunocytochemical evidence indicates that the majority of cells are cotransfected by all plasmids in triple transfection, our experimental condition did not allow for a tight control of the expression of NMDA receptor subunits. This produced the result that many cells were characterized by deactivation time course and haloperidol sensitivities of separate NR1/NR2A and NR1/NR2B subunit heteromers. We also speculate on the possible formation of channels resulting from the coassembly in the same receptor of NR1/NR2A/NR2B subunits from a minority of cells that gave responses to brief application of l-glutamate characterized by slow deactivation time course and decreased haloperidol sensitivity.

Plasticity of the developing visual system has been regarded as the best model for changes of neuronal connections under the influence of the environment. N -methyl-D-aspartate (NMDA) receptors are crucial for experience-dependent synaptic modifications that occur in the developing visual cortex. NMDA-mediated excitatory postsynaptic currents (EPSCs) in layer IV neurons of the visual cortex lasted longer in young rats than in adult rats, and the duration of the EPSCs became progressively shorter, in parallel with the developmental reduction in synaptic plasticity. This decrease in NMDA receptor-mediated EPSC duration is delayed when the animals are reared in the dark, a condition that prolongs developmental plasticity, and is prevented by treatment with tetrodotoxin, a procedure that inhibits neural activity. Application of L-glutamate to outside-out patches excised from layer IV neurons of young, but not of adult, rats activated prolonged bursts of NMDA channel openings. A modification of the NMDA receptor gating properties may therefore account for the age-dependent decline of visual cortical plasticity.

Pharmacologically isolated miniature NMDA receptor‐mediated excitatory postsynaptic currents (mN‐EPSCs) were recorded in large visual cortical neurons in layer V of rat cortical slices. Haloperidol (100 μm) and CP101,606 (10 μm), two specific blockers of NMDA receptors comprising NR1/NR2B subunits, were tested on mN‐EPSCs in rats at postnatal days 7 and 8 (P7–P8) and P13–P15. At both ages tested, no significant effects of these drugs were seen in the whole population of neurons, although in few neurons at both ages changes in amplitude were observed with haloperidol. Other dopamine receptor antagonists, spiperone and clozapine, failed to decrease mN‐EPSCs in cortical neurons at P13–P15. CP101,606 (10 μm) significantly decreased the amplitude of evoked N‐EPSCs (eN‐EPSCs) in visual cortical slices from rats at P3–P5, a developmental stage at which mRNA studies have indicated the virtual absence of NR2A mRNA. CP101,606 failed to significantly change evoked AMPA‐mediated EPSCs at P5 and eN‐EPSCs at P7–P8 and P13–P15. NMDA receptor‐mediated currents were also studied in somatic outside‐out patches at P13–P15 with fast application of l‐glutamate (1 mm). Haloperidol (50 μm) and CP101,606 (10 μm) blocked these currents in all patches tested. The effect of CP101,606 was concentration dependent. We suggest that rather early in development synaptic receptors comprising NR1/NR2B subunits could be associated with other subunits so that blockade by haloperidol and CP101,606 is prevented. Moreover, the consistent blockade seen in outside out patches might be ascribed to the confinement of NR1/NR2B receptors to an extrasynaptic population.