Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture

Journal Article

We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro. © 2002 Published by Elsevier Science Ltd.

Full Text

Cited Authors

  • Luo, JH; Fu, ZY; Losi, G; Kim, BG; Prybylowski, K; Vissel, B; Vicini, S

Published Date

  • 2002

Published In

Volume / Issue

  • 42 / 3

Start / End Page

  • 306 - 318

International Standard Serial Number (ISSN)

  • 0028-3908

Digital Object Identifier (DOI)

  • 10.1016/S0028-3908(01)00188-5