A novel two-site enzyme immunoassay reveals the regional distributions of and developmental changes in GluR1 and NMDAR1 protein contents in the rat brain

Glutamate receptors, including the alpha-amino-3-hydroxy-4-methylisoxazole- 4-propionic acid (AMPA) and NMDA receptors, play an important role in neura l development and synaptic plasticity in the brain. To date, it has been di fficult to correlate accurately individual biochemical phenomena with quant itative and qualitative changes in receptors occurring in specific neurons or synapses, In the present study, we established a two-site enzyme immunoa ssay for two key subunits of the AMPA and NMDA receptors. Its sensitivities were extremely high, 30 pg for GluR1 and 15 pg for the NMDAR1 receptor con taining the C2 exon [NMDAR1(C2)], which enabled us to measure their content s in a few milligrams of hippocampal tissue. Regional and developmental var iations in receptor protein levels were much more marked than those reporte d for mRNA: The absolute GluR1 protein content was highest in the rat hippo campus, whereas the NMDAR1(C2) content was high in all the forebrain region s examined. GluR1 protein levels increased most markedly during the second and third weeks of postnatal life, whereas NMDAR1(C2) content increased dur ing the first postnatal week. In the adult rat brain, the ratio of GluR1 pr otein to NMDAR1 protein was markedly lower in neocortical regions (similar to 2%) and the highest in cerebellum (22%). Therefore, this two-site enzyme immunoassay is a specific and unique method that enables us to measure abs olute tissue contents of the glutamate receptors and will lead to further i mportant discoveries on the biochemical alterations of these receptors.