The density and distribution of six GABAA receptor subunits in primary cultures of rat cerebellar granule cells

In cultured cerebellar granule neurons (seven daysin vitro) the expression of GABAA receptor subunits was quantified by using freeze-fracture immunocytochemical techniques with antibodies that specifically recognize the α1, α6, β2-3, γ2 and δ subunits of the GABAA receptor. In some experiments we have also used a less specific antibody that recognizes several α receptor subunits (α-total). The specificity of these antibodies was verified in human embryonic kidney cell line no. 293 cells transfected with complementary DNAs codifying for various GABAA receptor subunits. The most abundant labeling in granule cells was generated by the antibody against the β2-3 subunits (∼44 colloidal gold particles/μm2), while the specific antibodies against α1 and α6 subunits show a labeling of about 16 colloidal gold particles/μm2. The α-total antibody shows a labeling of ∼37 gold particles/μm2. Both the γ2 and δ antibodies show a labeling of about 10 gold particles/μm2. In granule cells, the relative proportion of the label density revealed with antibodies against α-total, β2-3, γ2 and δ subunits is approximately 4:4:1:1. Assuming that one molecular form of the α subunit is assembled in a GABAA receptor, it can be estimated that in granule cells about 50% of receptors include the α1 subunit. A similar relative abundance can be estimated for the α6 subunit. The proportion of GABAA receptors containing the γ2 or δ subunits can be estimated to be about 50% in each case. Cerebellar granule cells express various abundances of GABAA receptor subunits which can be estimated by freeze-fracture immunocytochemistry. Fifty to sixty percent of these subunits form small receptor clusters, which appear to be associated with neuronal cytoskeleton proteins. © 1995 IBRO.