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Abstract
An understanding of the N-methyl-D-aspartate (NMDA) receptor is crucial to understanding normal excitatory transmission in the mammalian central nervous system and to drug development for various diseases. Using immunoblotting, immunoprecipitations, and immunocytochemistry, the expression, localization, and interactions of NMDA receptor subunits were examined. Previous studies have suggested that NR1 subunit localization is determined by the splice variant form of NR1 present, and that NR2A and NR2B are selectively targeted to synaptic and extra-synaptic populations, respectively. The enrichment of these splice cassettes and subunits in rat cortical and cerebella postsynaptic density fractions was measured. No single cassette of NR1 was differentially enriched in the postsynaptic densities, and NR2A and NR2B subunits were similarly enriched at the synapse. Thus, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure. Using a rabbit polyclonal antibody developed in the laboratory to NR3A, NR3A protein expression was examined, and was found to peak between postnatal days 7 and 10 in the cortex, midbrain, and hippocampus, reaching higher maximal levels compared to the olfactory bulb and cerebellum. To examine the interactions of NR3A with the other NMDA receptor subunits, adult rat cortex was immunoprecipitated with an anti-NR1 antibody. Roughly 80% of NR3A was associated with NR1 in postnatal day 10 rat cortex, decreasing to half in the adult. Immunoprecipitating with the anti-NR3A antibody; roughly 5 to 10% of NR1, NR2A, and NR2B was associated with NR3A at postnatal day 10, decreasing by 50% in the adult. Thus, NR3A is expressed, distributed, and associated with other subunits, supporting its role in synaptic transmission. Finally, NR3A protein levels in normal and injured rat spinal cord tissue were measured. The level of NR3A was highest in P5 spinal cord and decreased to adult levels by P20. The hypothesis that NR3A expression is down-regulated following contusive SCI was tested by immunoblotting samples from laminectomy control and injured spinal cord samples. NR3A subunit expression decreased only in the cervical cord at 24 hours after SCI. Overall, these data confirm the high level of complexity of the NMDA receptor and the glutamatergic synapse.
