Colocalization of multiple GABA(A) receptor subtypes with gephyrin at postsynaptic sites

Clustering of gamma aminobutyric acid (GABA)(A) receptors to postsynaptic sites requires the presence of both the gamma2 subunit and gephyrin. Here, we analyzed by double-immunofluorescence staining the colocalization of gephyrin and major GABA(A)-receptor subtypes distinguished by the subunits alpha1, alpha2, alpha3, or gamma2 in adult rat brain. By using confocal laser scanning microscopy, GABA(A)-receptor subunit staining revealed brightly stained clusters that were colocalized with gephyrin-positive clusters of similar size and distribution in several brain regions, including cerebellum, hippocampus, thalamus, and olfactory bulb. In addition, a diffuse staining was observed for GABA(A)-receptor subunits in the neuropil, presumably representing extrasynaptic receptors. Overall, only few gephyrin-positive clusters were not colocalized with GABA(A)-receptor subunit clusters. Electron microscopic analysis in cerebellar cortex confirmed the selective postsynaptic localization of gephyrin. High-resolution images (voxel size, 50 x 50 x 150 nm) were restored with an iterative image deconvolution procedure based on a measured point-spread function to analyze the colocalization between GABA(A)-receptor subunits and gephyrin in individual clusters. This analysis revealed a considerable heterogeneity in the micro-organization of these presumptive GABAergic postsynaptic sites. For instance, whereas gephyrin- and gamma2 subunit-positive clusters largely overlapped in the cerebellar molecular layer, the colocalization was only partial in glomeruli of the granule cell layer, where small gephyrin clusters typically were "embedded" in larger GABA(A)-receptor clusters. These findings show that gephyrin is associated with a majority of GABA(A)-receptor subtypes in brain, and document the usefulness of image deconvolution for analyzing the structural organization of the postsynaptic apparatus by fluorescence microscopy.