Developmental changes in synaptic AMPA and NMDA receptor distribution and AMPA receptor subunit composition in living hippocampal neurons

AMPA and NMDA receptors mediate most excitatory synaptic transmission in th e CNS. We have developed antibodies that recognize all AMPA or all NMDA rec eptor variants on the surface of living neurons. AMPA receptor variants wer e identified with a polyclonal antibody recognizing the conserved extracell ular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibod y that binds to an extracellular N-terminal epitope of the NR1 subunit, com mon to all splice variants. In non-fixed brain sections these antibodies ga ve labeling patterns similar to autoradiographic distributions with particu larly high levels in the hippocampus. Using these antibodies, in conjunctio n with GluR2-specific and synaptophysin antibodies, we have directly locali zed and quantified surface-expressed native AMPA and NMDA receptors on cult ured living hippocampal neurons during development. Using a quantitative ce ll ELISA, a dramatic increase was observed in the surface expression of AMP A receptors, but not NMDA receptors, between 3 and 10 d in culture. Immunoc ytochemical analysis of hippocampal neurons between 3 and 20 d in vitro sho ws no change in the proportion of synapses expressing NMDA receptors (simil ar to 60%) but a dramatic increase (similar to 50%) in the proportion of th em that also express AMPA receptors. Furthermore, over this period the prop ortion of AMPA receptor-positive synapses expressing the GluR2 subunit incr eased from similar to 67 to similar to 96%. These changes will dramatically alter the functional properties of hippocampal synapses.