Somato-synaptic variation of GABA_A receptors in cultured murine cerebellar granule cells: investigation of the role of the α6 subunit

Abstract

Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA_A receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn²⁺ (150 μM), which depressed response amplitude by 82±5% and almost doubled the rate of channel deactivation. The GABA_A receptor α6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Δα6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA_A receptor function. All differences between cell body and synaptic GABA_A receptors observed in wild-type (WT) granule cells persisted in Δα6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the α6 subunit. However, mIPSCs from WT and Δα6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Δα6lacZ). This provides good evidence for a functional contribution of the α6 subunit to postsynaptic GABA_A receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Δα6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Δα6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Δα6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden, W., 1997. Ligand-gated ion channel partnerships: GABA_A receptor α₆ subunit inactivation inhibits δ subunit expression. Journal of Neuroscience 17, 1350–1362).

Introduction

GABA_A receptor function in phasic inhibitory synaptic communication is well established (Macdonald and Olsen, 1994). However, functional GABA_A 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 GABA_A 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) GABA_A receptors exhibit considerable diversity. This diversity presumably reflects fine tuning of the GABA_A receptor's function to the fulfilment of precise physiological roles.

GABA_A receptors are heteropentamers (Macdonald and Olsen, 1994, Sieghart, 1995). The kinetics, agonist/antagonist efficacy and pharmacology of recombinant GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A receptors on cultured cerebellar granule cells.