European Journal of Neuroscience, Vol. 11, pp. 1685–1697, 1999 © European Neuroscience Association Alterations in the expression of GABA A receptor subunits in cerebellar granule cells after the disruption of the α6 subunit gene Zoltan Nusser, 1, * Zahida Ahmad, 1 Verena Tretter, 2 Karoline Fuchs, 2 William Wisden, 3 Werner Sieghart 2 and Peter Somogyi 1 1 Medical Research Council Anatomical Neuropharmacology Unit, Mansfield Road, Oxford OX1 3TH, UK 2 Section of Biochemical Psychiatry, University Clinic for Psychiatry, Wahringer-Gurtel 18–20, A-1090 Vienna, Austria 3 MRC Laboratory of Molecular Biology, MRC Centre, Cambridge CB2 2QH, UK Keywords: immunocytochemistry, inhibition, ion channel, neurotransmission, synapse Abstract Any given subunit of the heteromultimeric type-A γ-aminobutyric acid (GABA) GABA A receptor may be present in several receptor subtypes expressed by individual neurons. Changes in the expression of a subunit may result in differential changes in the expression of other subunits depending on the subunit composition of the receptor subtype, leading to alterations in neuronal responsiveness to GABA. We used the targeted disruption of the α6 subunit gene to test for changes in the expression of other GABA A receptor subunits. Immunoprecipitation and ligand binding experiments indicated that GABA A receptors were reduced by μ 50% in the cerebellum of α6 –/– mice. Western blot experiments indicated that the α6 subunit protein completely disappeared from the cerebellum of α6 –/– mice, which resulted in the disappearance of the δ subunit from the plasma membrane of granule cells. The amount of β2, β3 and γ2 subunits was reduced by μ 50%, 20% and 40%, respectively, in the cerebella of α6 –/– mice. A comparison of the reduction in the level of α1, β2, β3, γ2, or δ-subunit-containing receptors in α6 –/– cerebellum with those observed after removal of α6-subunit-containing receptors from the cerebella of α6 +/+ mice by immuno-affinity chromatography demonstrated the presence of a significantly higher than expected proportion of receptors containing β3 subunits in α6 –/– mice. The receptors containing α1, β2, β3 and γ2 subunits were present in the plasma membrane of granule cells of α6 –/– mice at both synaptic and extrasynaptic sites, as shown by electron microscopic immunocytochemistry. Despite the changes, the α1 subunit content of Golgi-cell-to-granule-cell synapses in α6 –/– animals remained unaltered, as did the frequency of α1 immunopositive synapses in the glomeruli. Furthermore, no change was apparent in the expression of the α1, β2 and γ2 subunits in Purkinje cells and interneurons of the molecular layer. These results demonstrate that in α6 –/– mice, the cerebellum expresses only half of the number of GABA A receptors present in wild-type animals. Since these animals have no gross motor deficits, synaptic integration in granule cells is apparently maintained by α1-subunit-containing receptors with an altered overall subunit composition, and/or by changes in the expression of other ligand and voltage gated channels. Introduction A widely used approach to study the functional role of a protein in the CNS is to generate animals in which the expression of the protein is selectively disrupted. Although temporally and spatially restricted genetic deletions have already been developed (Kuhn et al., 1995; Tsien et al., 1996; Jones et al., 1997), it is often a general assumption that the expression of only a single gene is altered, without any change in the amount or in the precise subcellular location of other gene products. Inhibition in the brain is mainly mediated through type-A γ- aminobutyric acid (GABA) GABA A receptors, which are ligand- Correspondence: Any author, as above. E-mail: nusser@ucla.edu *Present address: Department of Neurology, UCLA School of Medicine, Los Angeles, California 90095–1769, USA Received 22 May 1998, revised 17 December 1998, accepted 22 December 1998 gated anion channels formed of pentameric assemblies of subunits. Although, the possible permutations of the 16 subunits to form pentameric channels are hundreds of thousands, it is generally accepted that only a limited number of receptor subtypes exists in the CNS (Sieghart, 1995; Stephenson, 1995; McKernan & Whiting, 1996; Mohler et al., 1996; Barnard et al., 1998). Cerebellar granule cells provide an excellent opportunity to study how the disruption of a gene for a single GABA A receptor subunit influences the amount and the precise subcellular location of other subunits, as these cells express six GABA A receptor subunits abundantly (Laurie et al., 1992; Persohn et al., 1992; Wisden et al., 1996). These subunits form GABA A receptor subtypes with distinct subunit compositions and distinct kinetic and pharmacological properties (Puia et al., 1994; Saxena & Macdonald, 1994, 1996; Kaneda et al., 1995; McKernan & Whiting, 1996; Tia et al., 1996b; Jechlinger et al., 1998). In addition, the microcircuit of the cerebellum is relatively simple; pre- and postsynaptic elements can easily be identified on ultrastructural grounds. For example, most granule cells receive GABAergic innerva-