Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat

Abstract

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of gamma-aminobutyric acid (GABA) and of two major GABA(A)-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the alpha1 and gamma2 subunits of the GABA(A) receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA(A)-receptor subtypes in the postsynaptic membrane.

Cross-talk and Co-trafficking between ρ1/GABA Receptors and ATP-gated Channels

“…Dynamic regulation of GABA A and GABA C receptor numbers at the membrane is regulated by interactions with several proteins. For example, gephyrin and dystrophin have been shown to play a role in the anchoring of GABA A receptors, although no physical interaction with gephyrin has been demonstrated in vitro (39,40). GABARAP, a microtubuleassociated protein, and Plic-1, an ubiquitin-related protein, interact with GABA A receptor subunits and are involved in receptor trafficking (41,42).…”

Differential Dependence of Axo-Dendritic and Axo-Somatic GABAergic Synapses on GABAAReceptors Containing the α1 Subunit in Purkinje Cells

“…Juvenile (P10 and P14; n ϭ 2 per genotype) and adult (n ϭ 5 per genotype) mice were perfused as above with a fixative additionally containing either 2% (fixative 1) or 0.1% (fixative 2) glutaraldehyde, and brains were postfixed overnight. Tissue blocks prepared with fixative 1 were washed in phosphate buffer, postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated in ethanol, and embedded in Epon 812, as described previously (Giustetto et al, 1998). Ultrathin sections were collected on nickel grids and labeled with antibodies to GABA and glutamate (Table 1) (Phend et al, 1992).…”

“…The sections were cryoprotected in 30% sucrose and repeatedly frozen and thawed to enhance antibody penetration. They were then collected in PBS and processed free-floating as described in detail previously (Giustetto et al, 1998). After incubation at room temperature in primary (72 h) ( Table 1) and secondary (goat antimouse conjugated to biotin, 1:250; Vector Laboratories, Burlingame, CA) antibodies, the sections were treated with 3,3Ј-diaminobenzidine, and the reaction product was silver-intensified and gold-toned.…”

Gephyrin: a central GABAergic synapse organizer

“…[28][29][30][31][32][33] Strikingly, the inhibitory synaptic localization of Figure 1 Schematic depiction of gephyrin domains (G, C and E) with sites for posttranslational modifications (phosphorylation and palmitoylation) and binding interactions with other synaptic proteins indicated. Gephyrin is phosphorylated at serine 268 and serine 270 by extracellular signal-regulated kinases 1 and 2 (ERK1/2) and glycogen synthase kinase-β (GSK-3β), respectively.…”