NMDA受体在培养海马神经元树突树上的表面表达及定位  

SURFACE EXPRESSION AND LOCALIZATION OF NMDARECEPTOR ON DENDRITIC TREE OF HIPPOCAMPAL NEURONS IN CULTURE

作　　者：傅展燕[1] 罗建红[1] 魏尔清[1] 

机构地区：[1]浙江大学医学院神经生物学实验室,杭州310031

出　　处：《解剖学报》2002年第4期410-414,共5页Acta Anatomica Sinica

基　　金：国家"973"计划基金 (G19990 5 40 0 3);国家自然科学基金资助项目 ( 39970 844)

摘　　要：目的　研究NMDA受体在不同发育阶段的培养大鼠海马神经元的表面表达以及在树突结构上的定位。　方法　构建绿荧光蛋白 (GFP)标记的NMDA受体NR1a亚单位的表达载体 (GFP NR1a) ,转染原代培养 5d的大鼠海马神经元 ,用抗GFP抗体和Cy3交联的二抗染色活细胞表面的受体簇。结合青荧光蛋白 (CFP)的共表达突出神经元的结构细节 ,观察表面NMDA受体簇的分布。　结果　GFP NR1a转染的神经元能表达点状、且分布于全细胞的绿荧光NMDA受体簇 ,在成熟神经元的树突上多为表面受体簇。培养 7d和培养 17d的转染神经元树突表面的NMDA受体簇密度并无显著差异。另外 ,培养 7d的海马神经元 ,表面NMDA受体簇几乎都位于树突干上 ,而树突丝上则罕见 ;培养 2周后 ,约一半的NMDA受体簇分布于树突棘。　结论　表面NMDA受体簇的分布具有树突结构相关的特异性 ,尤其是发现在发育早期NMDA受体簇罕见于树突丝 ,却已广泛分布于树突干上 ,且密度相当于成熟神经元。Objective To investigate the developmental profiles on surface expression and distribution of NMDA receptor clusters especially on dendritic structures in cultured hippocampal neurons of rats. Methods A vector expressing green fluorescent protein N-terminally tagged NR1a subunit(GFP-NR1a) was generated and transfected into cultured hippocamplal neurons at 5 days in vitro (DIV 5).Surface expressed GFP-NR1a containing NMDA receptor clusters was then labeled in living neurons by using anti-GFP 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(CFP). Results Punctate green flourescence clusters can be seen throughout the neurons transfected with GFP-NR1a and surface staining of living neurons showed that the most of these flourescence clusters on dendritic arbors were surface expressed in mature neurons.In parallel,no significant difference was found in terms of density of the surface NMDA clusters on dendritic trees in neurons at different developmental stage.Interestingly,at DIV 7,surface NMDA receptor clusters were mostly observed on the dendritic shafts,but rarely on filopodia.In contrast,two weeks later about 50% of clusters were found located at the dendritic spines.Conclusion This study shows that surface NMDA receptor clusters are distributed in a differentiated way related to dendritic structures during neural development.Especially at very early developmental stage,no clusters was found presented on filopodia,instead they were extensively clustered with a density estimated to be not significantly different from mature neurons.Therefore,our results suggested that NMDA receptors are likely to be incorporated or recruited to postsynaptic sites in the form of pre-expressed surface cluster on dendritic shafts during glutamatergic synapse formation.

关 键 词：NMDA受体 树突 海马神经元 绿荧光蛋白 发育 

分 类 号：R322.8[医药卫生—人体解剖和组织胚胎学]