Lévi S, Vannier C, Triller A
Laboratoire de Biologie Cellulaire de la Synapse (INSERM U 497), Ecole Normale Supérieure, Paris, France.
J Cell Sci. 1998 Feb;111 ( Pt 3):335-45. doi: 10.1242/jcs.111.3.335.
The cellular and molecular mechanisms underlying the postsynaptic aggregation of ionotropic receptors in the central nervous system are not understood. The glycine receptor (GlyR) and its cytoplasmic domain-associated protein, gephyrin, are clustered at the postsynaptic membrane and constitute a good model for addressing these questions. The glycine receptor is inhibited by strychnine. The effects of chronic strychnine treatment on the expression and cellular distribution of gephyrin and glycine receptor were therefore tested using primary cultures of spinal cord neurons. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the glycine receptor alpha1, alpha2, beta subunits and gephyrin mRNAs were expressed at comparable levels in strychnine-treated and untreated cultures. The number of immunoreactive cells and the subcellular distribution of gephyrin and GlyR subunits was determined with standard and confocal immunofluorescence. The proportion 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 nucleus. This cytoplasmic glycine receptor was not associated with the Golgi apparatus nor with the rough endoplasmic reticulum and therefore is not likely to correspond to neosynthesized proteins. The number of GlyR clusters on the somato-dendritic membrane was dramatically reduced on neurons displaying intracellular staining. In contrast, the subcellular distribution and the number of gephyrin clusters was not modified by the treatment. The fact that gephyrin postsynaptic localization was not modified by strychnine suggests that the aggregation of glycine receptor and gephyrin is governed by different mechanisms. The distribution of other cell surface molecules such as NCAM or GABAA receptor beta2/3 subunits was not modified by strychnine treatment. Chronic exposure of the cultures to tetrodotoxin did not affect gephyrin or glycine receptor cluster formation. Taken together, these results indicate that functional glycine receptor, but not electrical synaptic activity, is required for the formation of glycine receptor clusters.
中枢神经系统中离子型受体突触后聚集的细胞和分子机制尚不清楚。甘氨酸受体（GlyR）及其胞质结构域相关蛋白gephyrin聚集在突触后膜上，构成了研究这些问题的良好模型。甘氨酸受体受士的宁抑制。因此，利用脊髓神经元原代培养物测试了慢性士的宁处理对gephyrin和甘氨酸受体表达及细胞分布的影响。逆转录聚合酶链反应（RT-PCR）分析显示，在士的宁处理和未处理的培养物中，甘氨酸受体α1、α2、β亚基和gephyrin mRNA的表达水平相当。用标准免疫荧光和共聚焦免疫荧光法测定了免疫反应性细胞的数量以及gephyrin和GlyR亚基的亚细胞分布。士的宁处理不影响gephyrin和甘氨酸受体免疫反应性（IR）细胞的比例。共聚焦显微镜显示，甘氨酸受体主要定位于细胞核附近的细胞内。这种胞质甘氨酸受体与高尔基体和粗面内质网均无关联，因此不太可能对应新合成的蛋白质。在显示细胞内染色的神经元上，体树突膜上GlyR簇的数量显著减少。相比之下，gephyrin簇的亚细胞分布和数量并未因处理而改变。gephyrin突触后定位未因士的宁而改变这一事实表明，甘氨酸受体和gephyrin的聚集受不同机制调控。士的宁处理未改变其他细胞表面分子如神经细胞黏附分子（NCAM）或GABAA受体β2/3亚基的分布。培养物长期暴露于河豚毒素不影响gephyrin或甘氨酸受体簇的形成。综上所述，这些结果表明，甘氨酸受体簇的形成需要功能性甘氨酸受体，但不需要电突触活动。
