STRYCHNINE-SENSITIVE STABILIZATION OF POSTSYNAPTIC GLYCINE RECEPTOR CLUSTERS

The cellular and molecular mechanisms underlying the postsynaptic aggr egation of ionotropic receptors in the central nervous system are not understood, The glycine receptor (GlyR) and its cytoplasmic domain-ass ociated protein, gephyrin, are clustered at the postsynaptic membrane and constitute a good model for addressing these questions, The glycin e receptor is inhibited by strychnine, The effects of chronic strychni ne treatment on the expression and cellular distribution of gephyrin a nd glycine receptor were therefore tested using primary cultures of sp inal cord neurons, Reverse transcriptase-polymerase chain reaction (RT -PCR) analysis revealed that the glycine receptor alpha 1, alpha 2, be ta subunits and gephyrin mRNAs were expressed at comparable levels in strychnine-treated and untreated cultures. The number of immunoreactiv e cells and the subcellular distribution of gephyrin and GlyR subunits was determined with standard and confocal immunofluorescence. The pro portion of gephyrin and glycine receptor-immunoreactive (IR) cells was unaffected by strychnine treatment. Confocal microscopy revealed that the glycine receptor was mainly localized intracellularly near the nu cleus. This cytoplasmic glycine receptor was not associated with the G olgi apparatus nor with the rough endoplasmic reticulum and therefore is not likely to correspond to neosynthesized proteins, The number of GlyR clusters on the somatodendritic membrane was dramatically reduced on neurons intracellular staining, In contrast, the distribution and the number of gephyrin clusters was not modified by the treatment, The fact that gephyrin postsynaptic localization was not modified by stry chnine suggests that the aggregation of glycine receptor and gephyrin is governed by different mechanisms, The distribution of other cell su rface molecules such as NCAM or GABA(A) receptor beta 2/3 subunits was not modified by strychnine treatment, Chronic exposure of the culture s to tetrodotoxin did not affect gephyrin or glycine receptor cluster formation, Taken together, these results indicate that functional glyc ine receptor, but not electrical synaptic activity, is required for th e formation of glycine receptor clusters.