Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive nonN-methyl-Daspartate (non-NMDA) glutamate receptor antagonist [ 3H]6-cyano-7-nitro-quinoxaline-2,3-dione ( [3H]CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA 1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of ['HICNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 f 9.0 W a n d B,, = 3.56 k 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of ['HICNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of ['HIAMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KDI = 9.0 k 3.5 nM, B,,, = 0.15 k 0.05 pmol/mg protein, K,, = 278 k 50 W, and B,,, = 1.54 k 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of ~-['H]glutamate to quisqualate receptors and to sites labeled with ['HIAMPA. These results suggest that the non-NMDA glutamate receptor antagonist ['HICNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.