Somato-synaptic variation of GABAA receptors in cultured murine cerebellar granule cells: investigation of the role of the α6 subunit
Introduction
GABAA receptor function in phasic inhibitory synaptic communication is well established (Macdonald and Olsen, 1994). However, functional GABAA receptors are also located at extrasynaptic sites, including neuronal cell bodies. These receptors are thought to play roles in many other neuronal functions, including a tonic form of synaptic transmission found in post-migratory cerebellar granule cells (Brickley et al., 1996, Wall and Usowicz, 1997, Rossi and Hamann, 1998).
Characterisation of GABAA receptor-mediated responses in neurones from different brain regions, developmental stages and tissue culture conditions has revealed that the biophysics and pharmacology of both synaptic (Brickley et al., 1996, Frerking et al., 1995, Hollrigel and Soltesz, 1997, Mody et al., 1994, Tia et al., 1996a, Wall and Usowicz, 1997, Xiang et al., 1998) and extrasynaptic (Aguayo et al., 1994, Aguayo and Alarcon, 1993, Schonrock and Bormann, 1993, White, 1992, Zheng et al., 1994, Zhu et al., 1995, Xiang et al., 1998) GABAA receptors exhibit considerable diversity. This diversity presumably reflects fine tuning of the GABAA receptor's function to the fulfilment of precise physiological roles.
GABAA receptors are heteropentamers (Macdonald and Olsen, 1994, Sieghart, 1995). The kinetics, agonist/antagonist efficacy and pharmacology of recombinant GABAA receptors are strongly dependent on their subunit composition (Draguhn et al., 1990, Ebert et al., 1994, Gingrich et al., 1995, Hadingham et al., 1996, Lavoie et al., 1997, Pritchett et al., 1989, Saxena and Macdonald, 1994, Saxena and Macdonald, 1996, Smart et al., 1991, Tia et al., 1996a, Tia et al., 1996b, Verdoorn et al., 1990, Wafford et al., 1993). In addition, the biophysics and pharmacology of GABAA receptors are responsive to other factors such as phosphorylation (Gyenes et al., 1994, Jones and Westbrook, 1997a, Moss et al., 1992) and interaction with other proteins, for example those of the cytoskeleton (Kannenberg et al., 1997).
Phenotypic variation of native GABAA receptors is thought to arise, for the most part, from variations in underlying subunit composition caused by differential gene expression and subcellular targeting (Ebert et al., 1994, Gingrich et al., 1995, Hadingham et al., 1996, Lavoie et al., 1997, Pearce, 1993, Saxena and Macdonald, 1994, Saxena and Macdonald, 1996, Smart et al., 1991, Tia et al., 1996a, Tia et al., 1996b, Wafford et al., 1993, Wisden et al., 1996). Our previous work on cultured cerebellar granule cells points towards functional differences between cell body and synaptic GABAA receptors, particularly with respect to their kinetic properties (Mellor and Randall, 1998). In agreement with this, there is good histological evidence for segregation of different GABAA receptor subunits to the synaptic and extrasynaptic membrane of granule cells. For example the δ subunit is found only at the latter location (Nusser et al., 1998b).
The α6 subunit of the GABAA receptor is encoded by a developmentally regulated, highly conserved, cerebellar granule cell-specific gene (Bahn et al., 1996, Jones et al., 1997, Laurie et al., 1992, Varecka et al., 1994, Zheng et al., 1993). In vivo, its expression is first detectable around P5 and increases over the following 2–3 weeks of post-natal life (Mellor et al., 1998). Under appropriate conditions, α6 expression follows a similar developmental profile in vitro (Mellor et al., 1998). Although concentrated at postsynaptic sites (Nusser et al., 1996, Nusser et al., 1998b), the α6 subunit is also found on extrasynaptic membranes where it has been hypothesised to play a prominent role in spillover-mediated tonic neurotransmission (Rossi and Hamann, 1998, Nusser et al., 1998a, Nusser et al., 1998b). In expression systems, α6-containing recombinant GABAA receptors are benzodiazepine (BDZ)-insensitive and have been reported to exhibit a particularly high affinity for GABA (Zheng et al., 1994).
Mice lacking the α6 subunit of the GABAA receptor have been produced in two separate laboratories (Homanics et al., 1997, Jones et al., 1997). Although exhibiting normal behaviour under standard conditions, these animals exhibit increased behavioural sensitivity to BDZs (Korpi et al., 1998). By comparing granule cells from one of these mouse lines (Δα6lacZ, Jones et al., 1997) with wild-type animals, we have investigated the role the α6 subunit plays in determining the properties of GABAA receptors in cultured cerebellar granule cells. Our data indicate that this subunit contributes to the receptors responsible for the IPSC but is not responsible for the marked differences between somatic and synaptic GABAA receptors on cultured cerebellar granule cells.
Section snippets
Cell culture
Cultures were prepared from either wild type C57Bl6X129/Sv (WT) or homozygous Δα6lacZ mice (Jones et al., 1997). Post natal day 5 animals were killed under schedule 1 of UK Home Office regulations. The vermis and both hemispheres of the cerebellum were then dissected free and dissociated with trypsin to produce granule cell-rich cultures, as previously described (Mellor and Randall, 1997). Following plating onto matrigel-coated coverslips the cells were maintained in standard tissue culture
Differences in cell body and synaptic GABAA receptors revealed by response kinetics and Zn2+ sensitivity
Previous immunocytochemical and histological work in this laboratory has revealed that cerebellar cultures from WT mice are composed of a layer of GFAP-positive astrocytes underlying numerous interconnected neurofilament positive neurones (Leao et al., 2000). Most of the neurones in the culture have the classical spherical morphology of cultured granule cells. Interspersed between the granule cells, however, are a small number of more irregular shaped neurofilament-postive cells that seemingly
Discussion
Somatic and synaptic GABAA receptors in WT granule cells differ in their kinetics (Fig. 1), Zn2+ sensitivity (Fig. 2) and single channel conductance (Fig. 4). These differences persist in Δα6lacZ cells (Fig. 5, Fig. 6), thus ruling out the α6 subunit as their determinant. Whether the contrasting properties of cell-body and synaptic receptors in this system reflect the cellular location of different subunits or other factors, such as synapse specific phosphorylation or protein-protein
Acknowledgements
Supported by the MRC. ADR would like to thank Dr Mark Farrant for clarification of the details of his non-stationary noise analysis methods. JRM held an MRC PhD studentship for the duration of this work.
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Present address: Department of Cellular and Molecular Pharmacology, University of California San Francisco, 513 Parnassus Ave., San Francisco, USA.