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 expre ssion of GABA(A) receptor subunits was quantified by using freeze-frac ture immunocytochemical techniques with antibodies that specifically r ecognize 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 sp ecific antibody that recognizes several alpha receptor subunits (alpha -total). The specificity of these antibodies was verified in human emb ryonic kidney cell line no. 293 cells transfected with complementary D NAs codifying for various GABA(A) receptor subunits. The most abundant labeling in-granule cells was generated by the antibody against the b eta 2-3 subunits (similar to 44 colloidal gold particles/mu m(2)), whi le the specific antibodies against alpha 1 and alpha 6 subunits show a labeling of about 16 colloidal gold particles/mu m(2). The alpha-tota l antibody shows a labeling of similar to 37 gold particles/mu m(2). B oth 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 la bel density revealed with antibodies against alpha-total, beta 2-3, ga mma 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 inc lude the alpha 1 subunit. A similar relative abundance can be estimate d for the alpha 6 subunit. The proportion of GABA(A) receptors contain ing 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 immuno cytochemistry. Fifty to sixty percent of these subunits form small rec eptor clusters, which appear to be associated with neuronal cytoskelet on proteins.