Expression and heteromeric interactions of non-N-methyl-D-aspartate glutamate receptor subunits in the developing and adult cerebellum.

The localization and expression of ionotropic non-N-methyl-D-aspartate glutamate receptors (GluR) were investigated in the developing and adult rat cerebellum using subunit-specific polyclonal antibodies for immunocytochemical, immunoblot and immunoprecipitation studies. In P7 animals, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor immunoreactivity was detected in all layers of the cerebellar cortex with the exception of the external granule cell layer. Antibodies against the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR4 gave strong immunoreactive staining of Bergmann glia in both young and adult animals, and both antibodies showed prominent staining of the molecular layer in the adult cerebellum. Dense immunoreactive staining of Purkinje cell somata and dendrites was obtained with anti-GluR2/3/4c in both the developing and adult cerebellum. Whereas each of the three alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antibodies stained the internal, but not the external, granule cell layer, immunostaining for the kainate-type subunits GluR6/7 and KA2 was detected in both the external and internal granule cell layers. as well as in the molecular layer in both P7 and adult cerebellum. Immunoblot analysis of total cerebellar protein indicated that the level of GluR4 expression increased 15-fold from P1 to P18, whereas the expression of the KA2 subunit protein was nine-fold lower in adult cerebellum than it was at P1. The expression of GluR1 increased moderately (two-fold) from P1 to adult. Subunit interactions between GluR1 and GluR4, as well as between GluR6/7 and KA2, were demonstrated in immunoprecipitation experiments; and the GluR4 and KA2 subunits appear to be present exclusively in heteromeric assemblies with GluR1 and GluR6/7, respectively. The results show that the various alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type subunits are differentially expressed during cerebellar development and further define the possible subunit composition of non-N-methyl-D-aspartate receptors in the major cerebellar cell types.