NMDA受体与AMPA受体亚单位在培养海马神经元树突上的表达及共定位

目的 :研究含 NR2 B亚单位的 NMDA受体 (以下简称 NR2 B- NMDAR)与含 Glu R2亚单位的 AMPA受体 (以下简称 Glu R2 - AMPAR)在不同发育阶段的培养海马神经元树突上的表面表达及共定位。方法 :以 FL AG- NR2 B、GFP- Glu R2及 CFP- Actin的表达载体共转染原代培养 5 d的大鼠海马神经元 ,分别用抗 FL AG单克隆抗体和山羊抗鼠二抗 - Cy3(红色荧光 )、抗 GFP多克隆抗体和山羊抗兔二抗 - Alex4 88(绿色荧光 )作活细胞双重染色 ,显示并计数表面表达的 NR2 B- NMDAR受体簇、Glu R2 - AMPAR受体簇以及两者共定位的受体簇。结果 :培养 7d和 19d时 NR2 B- NMDAR受体簇密度分别为 39.7± 5 .0和 6 4.7± 6 .1(P<0 .0 1;n=6 ) ;Glu R2 - AMPAR受体簇密度分别为 5 9.1± 3.3和 99.7± 6 .4 (P<0 .0 1;n=6 ) ;两者共定位的受体簇密度分别为 2 9.9± 4 .5和 37.5± 2 .5 (P<0 .0 5 ;n=6 )。培养 7d和 19d时 ,与 Glu R2 - AMPAR共定位的 NR2 B- NMDAR受体簇占总 NR2 B- NMDAR受体簇的比例分别 75 .4 %和 5 7.9% ,而与 NR2 B- NMDAR共定位的 Glu R2 - AMPAR受体簇占总 Glu R2 - AMPAR受体簇的比例分别为 5 0 .6 %和 37.6 %。结论 :随着海马神经元的发育 ,成熟神经元比未成熟神经元树突表面 NR2 B- NMDAR受体簇、Glu R2 - A

Surface expression and co-localization of NMDA receptor and AMPA receptor on dendritic tree of hippocampal neurons in culture

OBJECTIVE: To investigate the developmental profiles on surface expression and co-localization of NMDA receptor clusters and AMPA receptor clusters on dendrite in cultured hippocampal neurons of rats. METHODS: Green fluorescent protein tagged GluR2 subunit (GFP-GluR2) and FLAG tagged NR2B subunit (FLAG-NR2B) were transfected into cultured hippocampal neurons at 5 days in vitro (DIV5). FLAG-NR2B containing NMDA receptor clusters and GFP-GluR2 containing AMPA receptor clusters expressed on membrane surface were then labeled in living neurons using anti FLAG mAb/Cy3-conjugated anti-mouse antibody and anti-GFP pAb/Alex488-conjugated anti-rabbit antibody. RESULT: The numbers of receptor cluster per 100 microm dendrite in the neurons at DIV7 and DIV19 were 39.7+/-5.0 and 64.7+/-6.1 (P<0.01) for NR2B-NMDAR, 59.1+/-3.3 and 99.7+/-6.4 (P<0.01) for GluR2-AMPAR, and 29.9+/-4.5 and 37.5+/-2.5(P<0.05) for the co-localized, respectively. At DIV7 and DIV19, 75.4% and 57.9% NR2B-NMDAR clusters were co-localized with GluR2-AMPAR; and 50.6% and 37.6% GluR2-AMPAR clusters were co-localized with NR2B-NMDAR, respectively. CONCLUSION: The density of NR2B-NMDAR containing and GluR2-AMPAR containing receptor clusters increases during development of hippocampal neurons in culture. Although the co-localized clusters are increased as well in an unit length of dendrite, the extent to which the two receptor clusters are co-localized decreases. These data imply a possible change in the partnership of AMPA receptor subtype and NMDA receptor subtype at newly formed synapses during development.