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 ad ult rat cerebellum using subunit-specific polyclonal antibodies for im munocytochemical, immunoblot and immunoprecipitation studies. In P7 an imals, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor immunoreactivity was detected in all layers of the cerebellar cortex w ith the exception of the external granule cell layer. Antibodies again st the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR4 gave strong immunoreactive staining of Bergma nn glia in both young and adult animals, and both antibodies showed pr ominent staining of the molecular layer in the adult cerebellum. Dense immunoreactive staining of Purkinje cell somata and dendrites was obt ained 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 exte rnal, granule cell layer, immunostaining for the kainate-type subunits GluR6/7 and KA2 was detected in both the external and internal granul e 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, w hereas the expression of the KA2 subunit protein was nine-fold lower i n adult cerebellum than it was at P1. The expression of GluR1 increase d moderately (two-fold) from P1 to adult. Subunit interactions between GluR1 and GluR4, as well as between GluR6/7 and KA2, were demonstrate d in immunoprecipitation experiments; and the GluR4 and KA2 subunits a ppear to be present exclusively in heteromeric assemblies with GluR1 a nd GluR6/7, respectively. The results show that the various alpha-amin o-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type subunits are differentially expressed during cerebellar development and furthe r define the possible subunit composition of non-N-methyl-D-aspartate receptors in the major cerebellar cell types. (C) 1997 IBRO. Published by Elsevier Science Ltd.