Cellular and sub-cellular localisation of GABA(B1) and GABA(B2) receptor proteins in the rat cerebellum

Following the recent discovery that GABA(B) receptors expressed in cell lin es are only functional when both GABA(B1) and GAB(B2) are expressed, the pr esent study reports on the development of polyclonal antisera specific for carboxyl-terminal portions of the two related GABA(B) receptor components r espectively. Western blotting indicated the specificity of affinity-purifie d antibodies for native or recombinant expressed GABA(Br1), and GABA(Br2), with no cross-reactivity, both antisera detecting the heterodimer in rat ce rebellar membranes. Immunohistochemistry revealed a distinct distribution o f both receptor proteins in rat cerebellum GABA(B1) immunoreactivity was pr imarily located in the granule cell layer and Purkinje cells, with discrete immuno-positive cell bodies being present in the molecular layer. GABA(B2) staining revealed intense immunoreactivity in thr molecular laypr with Wea ker Staining in the granule cell layer. Purkinje cell bodies were less inte nsely immune-positive for GABA(B2). Co-localisation of both receptor protei ns was observed using double immunofluorescence techniques, consistent with the notion that both proteins are required fur the formation of functional GABA(B) receptors in vivo. Immunofluorescence also indicated that GABA(B), receptors did not co-localise with glial fibrillary acid protein, confirmi ng a neuronal localisation for GABA receptors. Electron microscopic analysi s of the molecular layer revealed that the distribution of immunolabelling for both GABA(B1) and GABA(B2) was mainly located on the membrane of Purkin je cell dendrites and spines and in parallel fibre terminals. (C) 2000 Else vier Science B.V. All rights reserved.