SURFACE EXPRESSION AND COLOCALIZATION OF NMDA RECEPTOR AND AMPA RECEPTOR ON DENDRITE OF HIPPOCAMPAL NEURONS IN CULTURE / 解剖学报

ABSTRACT

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*!?m dendrite in DIV7 and DIV14 neurons were 59.2?5.6 and 74.8?3.1(P