Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons.

Each neuron in the mammalian brain carries many postsynaptic membrane specializations containing high densities of receptors that mediate signal transduction upon neurotransmitter release from the apposed nerve terminal. Little is known about the mechanisms by which receptors are transported to and anchored at postsynaptic sites, but extracellular as well as intracellular components may be involved. Ultrastructural studies have shown that the peripheral membrane protein gephyrin, which co-purifies with the postsynaptic inhibitory glycine receptor (GlyR) upon affinity chromatography, is situated on the cytoplasmic face of glycinergic postsynaptic membranes. Moreover, gephyrin binds with high affinity to polymerized tubulin and has been postulated to link the GlyR to the subsynaptic cytoskeleton. Here we report that treatment of rat spinal neurons in culture with gephyrin antisense oligonucleotides prevents the formation of GlyR clusters in the dendritic plasma membrane. Thus, gephyrin is essential for localizing the GlyR to presumptive postsynaptic plasma membrane specializations.

Distribution of gephyrin transcripts in the adult and developing rat brain.

The peripheral membrane protein gephyrin copurifies with the inhibitory glycine receptor of mammalian spinal cord. It binds with high affinity to polymerized tubulin and has been implicated in the anchoring of the glycine receptor to cytoskeletal elements. Recently, cDNA cloning has identified variants of the gephyrin mRNA, which originate from alternative splicing of four exonic regions (cassettes 1-4). In this study, the expression patterns of gephyrin splice variants were determined in the adult and developing rat brain by in situ hybridization with synthetic oligonucleotide probes. Gephyrin transcripts were detected throughout the brain and spinal cord, with mRNAs containing cassette 2 (C2 transcripts) being predominant in adult animals. C3 and C4 transcripts were seen in cerebellar granule cells and in the dentate gyrus, whereas a C1 probe did not produce detectable hybridization signals. During development, C2 and C3 mRNAs were found in most brain regions. Generally, the spatial and temporal distribution of gephyrin transcripts is similar to that of the glycine receptor beta subunit mRNA reported previously.

Modulation by NMDA Receptor Antagonists of Glycine Receptor Isoform Expression in Cultured Spinal Cord Neurons.

Two developmentally regulated isoforms of the inhibitory glycine receptor harbouring different alpha subunit variants, GlyRN (neonatal) and GlyRA (adult), have previously been identified in rodent spinal cord. Primary cultures of embyronic spinal neurons, however, express predominantly GlyRN. Here, N-methyl-d-aspartate (NMDA) receptor antagonists were found to significantly increase glycine receptor levels in mouse spinal cord cultures. In the presence of 2-amino-5-phosphonovalerate or MK-801 (dizocilpine), both GlyRN and GlyRA contents were elevated, as revealed by isoform-selective immunoassays and amplification of corresponding alpha subunit transcripts by the polymerase chain reaction. This effect of NMDA receptor antagonists was restricted to a 'sensitive' period within the second week after plating. Apparently, NMDA receptor-mediated glutamate neurotoxicity prevented GlyRA accumulation under standard culture conditions. Our data indicate that neuronal maturation in cell culture depends on conditions which minimize cell death resulting from glutamate release into the culture medium.