Cellular and sub-cellular localisation of GABAB1 and GABAB2 receptor proteins in the rat cerebellum

Following the recent discovery that GABA(B) receptors expressed in cell lines are only functional when both GABA(B1) and GAB(B2) are expressed, the present study reports on the development of polyclonal antisera specific for carboxyl-terminal portions of the two related GABA(B) receptor components respectively. Western blotting indicated the specificity of affinity-purified antibodies for native or recombinant expressed GABA(Br1), and GABA(Br2), with no cross-reactivity, both antisera detecting the heterodimer in rat cerebellar membranes. Immunohistochemistry revealed a distinct distribution of both receptor proteins in rat cerebellum GABA(B1) immunoreactivity was primarily 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 Weaker Staining in the granule cell layer. Purkinje cell bodies were less intensely immune-positive for GABA(B2). Co-localisation of both receptor proteins 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, confirming a neuronal localisation for GABA receptors. Electron microscopic analysis of the molecular layer revealed that the distribution of immunolabelling for both GABA(B1) and GABA(B2) was mainly located on the membrane of Purkinje cell dendrites and spines and in parallel fibre terminals. (C) 2000 Elsevier Science B.V. All rights reserved.