Autoradiographic Characterization Of L-(tritium)glutamate Binding Sites In The Central Nervous System (receptors, Nmda, Kainate, Quisqualate).

Abstract

A quantitative autoradiographic technique was developed to study L- ('3)H glutamate binding in sections of central nervous system tissue. This technique circumvented some problems associated with conventional receptor binding methodologies and allowed direct assessment of regional distribution, numbers and affinities of glutamate binding sites. The sensitivity and high degree of anatomical resolution attainable by autoradiography obviated the need for pooled samples of microdissected specimens. Under assay conditons, ('3)H glutamate bound rapidly and reversibly to sections of rat brain and was not metabolized appreciably. The distribution of glutamate binding sites corresponded to the projection areas of putative glutamatergic pathways. Thus, there was heavy glutamate binding in regions where there is evidence for glutamatergic innervation and little binding in nuclei which apparently do not receive glutamatergic input. Scatchard and Hill plots suggested that glutamate was interacting with a single population of sites; however, competition studies revealed binding site heterogeneity. One population of sites was sensitive to N-methyl-D-aspartate (NMDA) and other compounds specific for the NMDA receptor. This site was enriched in stratum radiatum of hippocampus (CA1) and represented a variable portion of glutamate binding sites elsewhere in the brain. A second population of sites had a high affinity for quisqualate. These sites were unevenly distributed and in the cerebellar molecular layer represented approximately 80% of glutamate binding sites. The number of high affinity quisqualate sites increased in the presence of chloride and calcium ions. A subset of high affinity quisqualate sites was sensitive to kainate, particularly in stratum lucidum of hippocampus (CA3). The density of the kainate-sensitive sites was decreased by calcium but not chloride ions. At high concentrations, quisqualate competed for all glutamate binding sites. There was a good correspondence between the density and distribution of low affinity quisqualate sites and NMDA-sensitive sites and pharmacological analysis suggested that these sites were equivalent. Anatomical and pharmacological evidence suggested that the NMDA-, high affinity quisqualate-, and kainate-sensitive glutamate binding sites may correspond to physiologically-defined NMDA, quisqualate and kainate receptors.