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GABAB receptor subunit expression in glia

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Abstract

GABAB receptor subunits are widely expressed on neurons throughout the CNS, at both pre- and postsynaptic sites, where they mediate the late, slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. The existence of functional GABAB receptors on nonneuronal cells has been reported previously, although the molecular composition of these receptors has not yet been described. Here we demonstrate for the first time, using immunohistochemistry the expression of GABAB1a, GABAB1b, and GABAB2 on nonneuronal cells of the rat CNS. All three principle GABAB receptor subunits were expressed on these cells irrespective of whether they had been cultured or found within brain tissue sections. At the ultrastructural level GABAB receptor subunits were expressed on astrocytic processes surrounding both symmetrical and assymetrical synapses in the CA1 subregion of the hippocampus. In addition, GABAB1a, GABAB1b, and GABAB2 receptor subunits were expressed on activated microglia in culture but were not found on myelin forming oligodendrocytes in the white matter of rat spinal cord. Together these data demonstrate that the obligate subunits of functional GABAB receptors are expressed in astrocytes and microglia in the rat CNS.

Introduction

γ-Amino butyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). GABAB receptors are functionally responsible for the late, slow component of GABA-mediated inhibitory synaptic transmission, which is insensitive to GABAA receptor antagonists (Hill and Bowery, 1981). GABAB receptors have been identified on both pre- and postsynaptic terminals. In the former instance GABAB receptors can exist as either auto- or heteroreceptors to modulate neurotransmitter release. GABAB receptors are members of the Type 3 (Class C) family of G-protein-coupled receptors (GPCRs) which are characterized by their large, extracellular amino terminal ligand binding domain. It is now widely believed that functional GABAB receptors are obligate heterodimers composed of two receptor subunits, GABAB1 and GABAB2, although functional homomeric receptors have been described Kaupmann et al 1997, Kaupmann et al 1998, Jones et al 1998, White et al 1998, Kuner et al 1999. That said, the existing dogma suggests that the GABAB1 receptor subunit is primarily responsible for agonist binding, whereas the GABAB2 receptor subunit is essential for trafficking of the heterodimer to the cell surface and signal transduction following agonist activation (see Couve et al., 2000; Calver et al., 2002).

Since the cloning of the GABAB1 and GABAB2 receptor subunits, and their associated splice variants, many studies have explored their distribution in the CNS and periphery. These reports have utilized a variety of techniques to localize each subunit, including autoradiography (Chu et al., 1990), in situ hybridization Bischoff et al 1999, Calver et al 2000, Liang et al 2000; Berthele et al., 2001), and immunohistochemistry Margeta-Mitrovic et al 1999, Sloviter et al 1999, Billinton et al 2000, Calver et al 2000, Ige et al 2000; Ng and Yung, 2001; Gonchar et al., 2001). Despite the differences in techniques and reagents used (e.g., different antisera) there has been general agreement between studies, in terms of the regional distribution of both GABAB1 and GABAB2 subunits, with a widespread distribution of both receptor subunits in a variety of neuronal populations throughout the brain and spinal cord.

In contrast, the expression of GABAB receptor subunits in nonneuronal cells has remained largely unexplored. GABAB immunoreactive (IR) cells in the white matter tracts of rat brain and spinal cord with GABAB antisera have been reported Margeta-Mitrovic et al 1999, Charles et al 2001. Binding of GABA to cultured astrocytes in the presence of unlabeled bicuculline has been demonstrated (Hosli and Hosli, 1990) and at the functional level, several reports have proposed roles for GABAB receptors in nonneuronal cells of the CNS, such as astrocytes. In particular Albrecht et al. (1986) demonstrated a reduction in [Ca2+] efflux in response to GABA or baclofen in rat cortical astrocyte cultures in the presence of the GABAA receptor antagonist bicuculline. Furthermore, baclofen but not the GABAA receptor agonist isoguvacine induced increases in intracellular Ca2+ (Nilsson et al., 1993) and inhibited the release of endogenous benzodiazepines from astrocytes (Patte et al., 1999).

Here, we now provide the first description of GABAB receptor subunit expression in glial cells of rodent brain. Using specific antisera we have assessed the receptor subunit expression profile on astrocytes, microglia, and oligodendrocytes at both the light and electron microscopic levels.

Section snippets

Expression of GABAB receptor subunits in astrocytes

GABAB receptor subunit expression on astrocytes in the hippocampus was assessed by colocalization with the astrocytic marker glial fibrillary acidic protein (GFAP). Colocalization was determined by a clear overlap of fluorescent GFAP- and GABAB-specific IR signals. GFAP-positive astrocytes were clearly labeled throughout the rat brain in regions including the hippocampus, cortex, cerebellum, and spinal cord. GABAB1a expression was observed in these areas on neurons as well as on cells

Discussion

In this report we have demonstrated for the first time that both GABAB1 and GABAB2 receptor subunits are expressed on astrocytes and microglia but not on oligodendrocytes. Over the past 15 years our understanding of glia in the CNS has dramatically changed. Glia are no longer thought to play a minor functional role in the CNS and are now considered critical for supporting neuronal function in the adult and developing brain, providing structural frameworks, contributing to the immune system of

Tissue preparation

Male Sprague–Dawley rats were anesthetized with sodium pentobarbital and transcardially perfused with heparinized saline followed by 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were removed and postfixed in 4% PFA for 24 h before transfer to 30% sucrose for 48 h at 4°C. Tissues were frozen in isopentane at −40°C and brain and spinal cord sections were cut at approximately −20°C using a cryostat (Leica, Milton Keynes, UK). All procedures involving experimental animals were conducted in

Acknowledgements

We thank D. Mitchell for assistance in producing primary hippocampal cell cultures.

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