Surface Expression And Colocalization Of NMDA Recptor And AMPA Receptor On Dendrites Of Cultured Hippocampal Neurons

A central assumption in neurobiology holds that synapse formation and synaptic plasticity underlie the creation of functional neural circuits and nervous system plasticity. Two major subtypes of ionotropic glutamate receptors(GluRs), N-methyl-D-aspartate (NMDA)-type and α-amino-3-hydroxy-5-methyl -4-isoxazolepropionate (AMPA) -type receptors, are found localized on glutamatergic postsynaptic excitatory synapse. Three gene families that encode NMDA receptor subunits have been identified: NR1, NR2A, and NR3 subunits. AMPA receptor subunits have been identified: GuR1 , GluR2, GluR3, GluR4 subunits.In our study, two vectors expressing green fluorescent protein N-terminally tagged GluR1 subunit (GFP-GluR1)/GluR2 subunit (GFP-GluR2) and FLAG N-terminally tagged NR2B subunit (FLAG-NR2B) were generated and transfected into cultured hippocampal neurons at 5 days in vitro (DIV5). Surface expressed FLAG-NR2B containing NMDA receptor clusters and GFP-GluR1 containing AMPA receptor clusters /GFP-GluR2 containing AMPA receptor clusters were then labeled in living neurons by using anti-FLAG primary antibody followed by Cy3-conjugated secondary antibody and anti-GFP primary antibody followed byAlex488-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).We find thatrl) Significant difference was found both in NR2B subunits containing NMDA receptor and GluRl subunits containing AMPA receptor between DIV7 and DFV14. Colocalization fraction of NR2B subunits containing NMDA receptor and that of GluRl subunits containing AMPA receptor were both increasing with the development of neurons. In mature neurons, 70% receptor clusters located at the dendritic spines were in the form of colocalization, and 40% overlaid clusters were located at the dendritic spines. 2) Density of GluR2 subunits containing AMPA receptor (GluR2-AMPAR)were increasing with the development of neurons. But the lever of increase was different (GluR2-AMPAR>NR2B-NMDAR >colocalization), however, Colocalization fraction of NR2B-NMDAR and GluR2-AMPAR were declining with the development of neurons.In a word, we find that during development of hippocampal neurons, the density of colocalization puncta between surface NR2B-NMDAR and GluRl-AMPAR ,or GluR2-AMPAR cluster on dendritic spines increases ,which suggest that more active synapses are formed , While the cooperation of NMDA receptor subunits and AMPA receptor subunits was heterogeneous, for the percentage of colocalizationof NR2B-NMDAR clusters with GluRl-AMPAR clusters increases, however,the percentage of colocalization of NR2B-NMDAR clusters with GluR2-AMPAR cluster decrease.