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

Abstract

Following the recent discovery that GABA B receptors expressed in cell lines are only functional when both GABA B1 and GABA 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 the molecular layer, with weaker staining in the granule cell layer. Purkinje cell bodies were less intensely immuno-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 for 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 B 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.