Colocalization of gephyrin and GABA_A‐receptor subunits in the rat retina

Abstract

Gephyrin is a protein that copurifies with the glycine receptor (GlyR) and is required for the clustering of GlyRs at postsynaptic sites. Previously, it was thought that antibody mAb 7a, directed against gephyrin, was a specific marker for GlyR. However, there is evidence that gephyrin can also be found at nonglycinergic synapses. Here, immunocytochemistry was applied to show this directly for the rat retina. Both gephyrin and different subunits of the gamma-aminobutyric acid (GABA)A receptor were localized to discrete puncta in the inner plexiform layer, and these puncta were shown by electron microscopy to represent synaptic sites. Double immunocytochemistry revealed that GABAA receptors and GlyRs are not colocalized. However, gephyrin and different subunits of GABAA receptors were found to occur at the same synapses. The amount of colocalization varied with the GABAA receptor subunit composition and was most extensive for the alpha 2 subunit, less for the alpha 3 subunit, and minimal for the alpha 1 subunit. The gephyrin present at GABAergic synapses of the retina might also be involved with clustering of receptors at the postsynaptic sites. Hence, localization of gephyrin can no longer be considered as a unique marker of glycinergic synapses.

Molecular and functional heterogeneity of GABAergic synapses

“…By immunofluorescence staining and immunoelectron microscopy in brain sections, gephyrin is selectively detected at postsynaptic sites of glycinergic and most GABAergic synapses, forming puncta (clusters) that are co-localized with GlyRs and GABA A Rs, respectively [62][63][64][65]. Initial observations of a functional interaction between gephyrin and GABA A R were made in γ2-KO mice, in which postsynaptic clustering of GABA A R and gephyrin was strongly impaired compared to wild-type and heterozygous littermates [66][67][68].…”

The expression of GABAAreceptors during the development of the rabbit retina

“…It is comprised of at least 13 subunits, which can be grouped by the degree of sequence homology into six different alpha subunits, three beta subunits, three gamma subunits, one delta and one delta subunits (Cutting et al 1991;Mö hler et al 1990Mö hler et al , 1995Laurie et al 1992;review: Mö hler et al 1997). These different subunits have different locations and properties in the retina (Enz et al 1995;Brandstätter et al 1995;Greferath et al 1995;Sassoë-Pognetto et al 1995;Ehinger & Zucker, 1996;Zucker & Ehinger, 1998). GABA A receptors are ligand gated ion channels in which the GABA binding site is directly linked to a chloride channel, but it is not yet well understood what these differences mean from a functional point of view (Feigenspan et al 1993).…”

“…However, good monoclonal antibodies for some of the most common GABA A receptors are available (the alpha 1 and beta 2/3 subunits; monoclonal antibodies bd24 and bd17) and they are likely to be useful also for studies on the developing retina. In adult animals it has been shown that they stain predominantly in the inner plexiform layer together with cells in the inner nuclear layer, and also some structures in the outer plexiform layer (Greferath et al 1993(Greferath et al , 1994Brandstätter et al 1995;Sassoë-Pognetto et al 1995;Ehinger & Zucker, 1996). However, their distribution in the developing rabbit retina is not well known.…”

“…Although the GABAergic neuron system and the GABA receptors have been characterized relatively well in the adult rabbit retina (Greferath 1993(Greferath , 1994Brandstätter et al 1995;Sassoë-Pognetto et al 1995;Ehinger & Zucker, 1996;Zucker & Ehinger, 1998), little is known about the function of GABA A receptors during different stages of the development of the retina or when they emerge. We have therefore examined the distribution and localization of GABA A receptors during the development of the rabbit retina, aiming at a better understanding of what role GABA might have in the regulation of the development of the tissue.…”

Molecular architecture of glycinergic synapses

“…Although punctate gephyrin immunoreactivity (IR) co-distributes with GlyR IR in those regions of the CNS, where GlyRs are expressed, gephyrin expression was shown to exceed by far that of GlyRs in the CNS (Kirsch and Betz 1993). Detailed analyses of brain regions devoid of GlyR expression revealed co-localization of punctate gephyrin IR with GABA A receptors, namely those harboring 2 and/or 2 subunits (Sassoe-Pognetto et al 1995). These Wndings were impressively conWrmed by analyzing GABA A receptor expression in gephyrin deWcient mice (Fischer et al 2000;Kneussel et al 1999a).…”