N-甲基-D-门冬氨酸受体与使君子酸受体在培养海马神经元树突上的膜表面表达与共定位  

SURFACE EXPRESSION AND COLOCALIZATION OF NMDA RECEPTOR AND AMPA RECEPTOR ON DENDRITE OF HIPPOCAMPAL NEURONS IN CULTURE

作　　者：邱爽[1] 王敏珍[2] 傅展燕[1] 黄曼[1] 罗建红[1] 

机构地区：[1]浙江大学医学院神经生物教研室 [2]浙江大学医学院邵逸夫医院妇产科,杭州310006

出　　处：《解剖学报》2004年第1期30-34,共5页Acta Anatomica Sinica

基　　金：国家"973"计划基金资助项目 (G19990 5 40 0 3 ) ;国家自然科学基金资助项目 ( 3 9970 844 ;3 0 2 70 43 6)

摘　　要：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 developmental profiles on surface expression and colocalization of NMDA receptor and AMPA receptor on dendritic structures in cultured hippocampal neurons of rats. Methods Two vectors expressing green fluorescent protein tagged GluR1 subunit(GFP-GluR1) and FLAG tagged NR2B subunit(FLAG-NR2B) were co-transfected into cultured hippocampal neurons at 5 days in vitro(DIV5).Surface expressed GFP-GluR1 containing AMPA receptor clusters and FLAG-NR2B containing NMDA receptor clusters were then labeled in living neurons by using anti-GFP primary antibody followed by Alex488-conjugated secondary antibody,and anti-FLAG primary antibody followed by Cy3-conjugated secondary antibody.Furthermore,distribution of the surface clusters was observed on the detailed dendritic structures visualized with co-expression of cyan fluorescent protein tagged actin(CFP-Actin). Results The numbers for GluR1 containing AMPA receptor clusters,NR2B containing NMDA receptor clusters,and colocalized receptor clusters per 100*!μm dendrite in DIV7 and DIV14 neurons were 59.2±5.6 and 74.8±3.1(P<0.05),38.7±3.5 and 80.8±4.9(P<0.01),and 16.3±5.2 and 40.1±6.0(P<0.01),respectively.In DIV14 neurons,40 percent of the coloclized receptor clusters were located at the dendritic spines,and 70 percent of receptor clusters located at the dendritic spines were in the form of colocalization.Conclusion This study indicates that the density of GluR1 containing AMPA receptor, NR2B containing NMDA receptor,and their colocalization are remarkably increased during postnatal development of hippocampal neurons in culture,and the colocalized clusters of NR2B containing NMDA receptor and GluR1 containing AMPA receptor are predominantly located at the dendritic spines in DIV14 neurons.

关 键 词：N-甲基-D-门冬氨酸受体 使君子酸受体 培养 海马 神经元树突上 膜表面表达 共定位 

分 类 号：Q189[生物学—神经生物学]