SURFACE EXPRESSION AND LOCALIZATION OF NMDARECEPTOR ON DENDRITIC TREE OF HIPPOCAMPAL NEURONS IN CULTURE / 解剖学报

ABSTRACT

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.