Disruption of postsynaptic GABAA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

We have used RNA interference (RNAi) to knock down the expression of the gamma 2 subunit of the GABA(A) receptors (GABA(A)Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the gamma 2 subunit, one targeting the coding region and the other one the 3'-untranslated region (UTR) of the gamma 2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA(A) receptor gamma 2 subunit and the clustering of other GABA(A)R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the gamma 2 shRNAs had no effect on the clustering of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A gamma 2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3'-UTR targeted by gamma 2 RNAi, rescued both the postsynaptic clustering of GABA(A)Rs and the GABAergic innervation. Decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the gamma 2 shRNAs. The results indicate that the postsynaptic clustering of GABA(A)Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.