THE DENSITY AND DISTRIBUTION OF 6 GABA(A) RECEPTOR SUBUNITS IN PRIMARY CULTURES OF RAT CEREBELLAR GRANULE CELLS

In cultured cerebellar granule neurons (seven days in vitro) the expression of GABA(A) receptor subunits was quantified by using freeze-fracture immunocytochemical techniques with antibodies that specifically recognize the alpha 1, alpha 6, beta 2-3, gamma 2 and delta subunits of the GABA(A) receptor. In some experiments we have also used a less specific antibody that recognizes several alpha receptor subunits (alpha-total). The specificity of these antibodies was verified in human embryonic kidney cell line no. 293 cells transfected with complementary DNAs codifying for various GABA(A) receptor subunits. The most abundant labeling in-granule cells was generated by the antibody against the beta 2-3 subunits (similar to 44 colloidal gold particles/mu m(2)), while the specific antibodies against alpha 1 and alpha 6 subunits show a labeling of about 16 colloidal gold particles/mu m(2). The alpha-total antibody shows a labeling of similar to 37 gold particles/mu m(2). Both the gamma 2 and delta antibodies show a labeling of about 10 gold particles/mu m(2). In granule cells, the relative proportion of the label density revealed with antibodies against alpha-total, beta 2-3, gamma 2 and delta subunits is approximately 4:4:1:1.. Assuming that one molecular form of the alpha subunit is assembled in a GABA(A) receptor, it can be estimated that in granule cells about 50% of receptors include the alpha 1 subunit. A similar relative abundance can be estimated for the alpha 6 subunit. The proportion of GABA(A) receptors containing the gamma 2 or delta subunits can be estimated to be about 50% in each case. Cerebellar granule cells express various abundances of GABA(A) 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.