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目的 研究含NR2B亚单位的N 甲基 D 门冬氨酸 (NMDA)受体 (NR2B NMDAR)与含GluR1亚单位的使君子酸 (AMPA)受体 (GluR1 AMPAR)在发育不同阶段的培养海马神经元树突上的膜表面表达与共定位。 方法 以FLAG NR2B和GFP GluR1表达载体共转染原代培养第 5d(DIV5 )的大鼠海马神经元 ,分别用相应特异性抗体和荧光标记二抗作活细胞染色 ,显示膜表面表达的受体簇。 结果 对DIV7和DIV14的培养海马神经元树突表面受体簇计数 (个数 10 0 μm) ,GluR1 AMPAR受体簇分别为 5 9 2± 5 6和 74 8± 3 1(P <0 0 5 ) ;NR2B NMDAR为 38 7±3 5和 80 8± 4 9(P <0 0 1) ;两者共定位为 16 3± 5 2和 4 0 1± 6 0 (P <0 0 1)。DIV14海马神经元 4 0 %的共定位受体簇分布在树突棘上。 结论 随着海马神经元的发育 ,树突膜表面NR2B NMDAR、GluR1 AMPAR及两者共定位受体簇密度均增加 ,提示 ,形成更多具有活性的兴奋性突触 ,且主要位于树突棘上。
Objective To investigate the effects of N-methyl D aspartate (NMDA receptor) (NR2B NMDAR) containing NR2B subunit and GluR1 AMPAR receptor (GluR1 AMPAR) containing subunits of GluR1 in culturing hippocampal neurons Membrane surface expression and co-localization on the dendrites. Methods The hippocampal neurons of the rat primary cultured for 5 days (DIV5) were cotransfected with FLAG NR2B and GFP GluR1 expression vectors, respectively. The corresponding specific antibodies and fluorescent secondary antibodies were used to stain the living cells, which showed that the receptors on the membrane surface cluster. Results The numbers of dendritic surface receptor clusters in hippocampal neurons cultured in DIV7 and DIV14 were counted (number of 10 0 μm), GluR1 AMPAR receptor clusters were 592 ± 5 6 and 74 8 ± 3 1 (P <0.05) ). NR2B NMDAR was 38 7 ± 3 5 and 80 8 ± 49 (P <0.01). The co-localization was 16 3 ± 5 2 and 4 0 1 ± 60 (P 0 01). 40% of DIV14 hippocampal neurons co-localized receptor clusters distributed on dendritic spines. Conclusion With the development of neurons in hippocampus, the density of NR2B NMDAR, GluR1 AMPAR on the surface of the dendritic membrane and their co-localization increased, suggesting that more active excitatory synapses are formed and located mainly on dendritic spines .