Elsevier

Neuropharmacology

Volume 42, Issue 1, January 2002, Pages 34-47
Neuropharmacology

Pharmacological heterogeneity of γ-aminobutyric acid receptors during development suggests distinct classes of rat cerebellar granule cells in situ

https://doi.org/10.1016/S0028-3908(01)00158-7Get rights and content

Abstract

The γ-aminobutyric acid receptor (GABAAR) represents a ligand-gated Cl-channel assembling as heteropentamere from 19 known subunits. Cerebellar granule cells contain a unique subset, namely the α1-, α6-, β2-, γ2- and δ-subunits. We studied their GABAergic pharmacology in situ using whole-cell patch-clamp recordings in brain slices and a modified Y-tube application system. The distribution of the EC50s for GABA in young (P8–P14) and medium aged animals (P15–P28) could be fitted with the sum of two Gaussian distributions with means of 60 and 185 μM and 27 and 214 μM, respectively. In older animals (P29–P48) the observed homogeneous range of sensitivities fitted a single Gaussian distribution (11 μM). In young animals (≤P14) GABA-responses were largely insensitive towards 300 μM of the α6-specific inhibitor furosemide (82% of control response). The sensitivity increased in older animals at the EC5–20 of GABA (31% of control responses), supporting an increased expression of α6-subunits as molecular basis for the observed developmental changes. Approximately 50% of cells in the age range P15–P48 were potentiated by 1 μM diazepam and by 3 μM methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), suggesting the concurrent presence of α1- and α6-containing receptors, whereas the remaining of cells were neither potentiated by diazepam nor did they show the α6-typical DMCM potentiation, though they were potentiated by loreclezole. These properties indicate unknown pharmacological characteristics of cerebellar receptor-subunit combinations in approximately 50% of granule cells in situ.

Introduction

γ-aminobutyric acid (GABA) is a ubiquitous neurotransmitter in the vertebrate central nervous system (CNS; Sivilotti and Nistri, 1991). It mediates fast synaptic inhibition through a ligand-gated chloride-channel, the GABAA receptor (GABAAR; Sakmann et al., 1983, Bormann and Clapham, 1985). In mammalia 19 subunits of this receptor are presently known and grouped into the eight classes α (1–6), β (1–3), γ (1–3), δ, ρ (1–3), ϵ, π, and θ (Davies et al., 1997, Barnard et al., 1998, Hevers and Lüddens, 1998, Bonnert et al., 1999, Neelands et al., 1999, Neelands and Macdonald, 1999). Their supposedly heteropentameric assembly (Nayeem et al., 1994) with a presumed stoichiometry of αx2βx2γx (Chang et al., 1996, Tretter et al., 1997) allows for a large heterogeneity of the ionotropic receptor complexes. The physiological significance of this heterogeneity remains poorly understood, partly due to our lack of understanding of native receptor compositions and their characteristics, the latter mainly derived from receptors expressed in heterologous expression systems.

The granule cells of the cerebellum express 10 different GABAAR-subunits during development (Wisden et al., 1996). After migration from the external to the inner granule cell layer during the first postnatal weeks an untypical combination of GABAAR-subunits can be found. Immunohistochemistry and mRNA expression (Laurie et al., 1992, Gao and Fritschy, 1995) indicate the spatially restricted α6- and δ-subunits as major receptor components in these cells in addition to the ubiquitously found α1-, β2/3- and γ2-subunits. The α6-subunit is almost confined to the cerebellar granule cells (Lüddens et al., 1990) and the δ-subunit is colocalised with either α6 in the cerebellum or with α4 in the thalamus and hippocampus (Wisden et al., 1992). Both, α6 and δ, are detected in increasing amounts following postnatal day six and twelve (P6, P12), respectively (Laurie et al., 1992, Montpied et al., 1998; for α6 in mice see: Varecka et al., 1994, Mellor et al., 1998). When expressed in HEK 293 cells together with β2/3- and γ2-, the α6-subunits confer high sensitivity to GABA with the δ-subunits further enhancing this sensitivity (Jackel et al., 1998, Hevers et al., 2000) and slowing down the apparent kinetics of recombinant receptors due to a reduced desensitisation (Haas and Macdonald, 1999). The α6-subunits also confer a unique sensitivity towards the diuretic furosemide (Korpi et al., 1995, Knoflach et al., 1996, Jackel et al., 1998) and an insensitivity towards the classical benzodiazepine (BZ) diazepam (Lüddens et al., 1990), though a sensitivity towards some partial BZ-modulators is maintained (Lüddens et al., 1990, Kleingoor et al., 1991). So is bretazenil still potentiating recombinant α6β2γ2 receptors (Knoflach et al., 1996), whereas RO15-1788 (flumazenil), RO15-4513 and the β-carboline methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), known as null- and negative modulators, respectively, potentiate GABA-responses in α6-containing receptors in contrast to their effects on α1-containing receptors (Knoflach et al., 1996, Saxena and Macdonald, 1996, Hevers et al., 2000; Szegedi et al., 2001).

The restricted expression of α6- and δ-containing receptors during postnatal development suggests specific functions of these subunits. Due to their high GABA-sensitivity and slowly desensitising characteristics in vitro these receptors have been suggested as extrasynaptic targets of GABA overspilling from nearby synapses (Wall and Usowicz, 1997, Nusser et al., 1998, Rossi and Hamann, 1998, Brickley et al., 2001). Their unusual pharmacological properties towards furosemide and the BZ-ligands indicated that they can be differentiated in functional cerebellar receptor subtypes in situ, but so far, the identity of specific receptor subtypes formed by the five major subunits α1, α6, β2/3, γ2, and δ has only been addressed by immunoprecipitation studies. The heterogeneity of the native receptor pool and the pharmacological diversity within single cells as well as the developmental changes during postnatal development as implied by the altered mRNA expression, have rarely been addressed in situ. With the exemption of a shift from furosemide-insensitive to increasingly furosemide-sensitive inhibitory postsynaptic currents (IPSCs) from P8 towards P29 (Tia et al., 1996), most developmental studies were restricted to single stages, e.g. single channel and fluctuation analysis of IPSCs in young rats (P7, pre- and postmigratory cells; Brickley et al., 1999; see also Mellor et al., 2000). Other approaches employed cell-culture systems in which the expression of α6- and δ-subunits might be differentially influenced by depolarisation (Zhu et al., 1995, Gault and Siegel, 1997, Gault and Siegel, 1998, Mellor et al., 1998). To study the developmental heterogeneity of GABAARs we modified a Y-tube system for focal drug application in upright slice recordings and characterised granule cells in situ using whole-cell patch-clamp recordings. We found a pronounced developmental shift from low to high GABA-sensitivity (EC50) with a two week transitional period (postnatal day 14 to 28) during which low and high sensitive cells coexisted in parallel. During the same time period the α6-specific furosemide inhibition increased. But whereas the simultaneous presence of furosemide- and diazepam-sensitivity indicated a parallel presence of α1- and α6- containing receptors in 50% of cells in animals older than P14, the pharmacological characteristics in the remaining 50% of cells indicate novel pharmacological properties of the native receptor subtypes.

Section snippets

Tissue preparation

Sprague Dawley rats were reared in the departmental animal facility and transferred to the local experimental room one to three hours prior to experiments. All animal procedures followed the German guidelines for animal care. Male rats between postnatal day 8 (P8) and day 48 (P48) were decapitated and their brains rapidly removed into ice-cold, carboxygenated (5% CO2/95% O2) artificial cerebrospinal fluid (ACSF, see Section 2.3). The cerebellar vermis was trimmed, glued to the stage of a 752M

General properties of granule cell responses

Using animals aged from P8 to P48, we recorded from cells in the inner granule cell layer of lobes VIII to X of the cerebellar vermis. In mice α6-subunit expression starts at these positions within the first weeks of postnatal cerebellar development (Mellor et al., 1998). The identity of granule cells, visually indicated by size and appearance, was verified by their electrical properties, e.g. small cell capacitances and high input resistances. The latter decreased with age from 8.1 GΩ in young

Discussion

The GABAergic characteristics of native cerebellar granule cells are expected to be heterogeneous due to the contribution of several different receptor populations. According to data obtained by immunoprecipitation the predominant α1-, α6-, β2/3, γ2 and δ-subunits assemble in these cells into ternary α1β2/3γ2, α6β2/3γ2, and α6β2/3δ and quaternary α1α6β2/3γ2 and α1α6β2/3δ receptors (Jechlinger et al., 1998). As IPSC recordings and nucleated patches are restricted to either synaptic or somatic

Acknowledgements

We thank E. Korpi and H. Rabe for critical comments on the manuscript. This work was supported by the ‘Stiftung für Innovation Rheinland-Pfalz’ and the Deutsche Forschungsgemeinschaft (Be 454/4-3).

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