Distribution, density, and clustering of functional glutamate receptors before and after synaptogenesis in hippocampal neurons

Postsynaptic differentiation during glutamatergic synapse formation is poor ly understood. Using a novel biophysical approach, we have investigated the distribution and density of functional glutamate receptors and characteriz ed their clustering during synaptogenesis in cultured hippocampal neurons. We found that functional alpha- amino-3-hydroxy-5-methyl-4-isoxazolpropiona te (AMPA) and N-methyl-D-aspartate (NMDA) receptors are evenly distributed in the dendritic membrane before synaptogenesis with an estimated density o f 3 receptors/mu m(2). Following synaptogenesis, functional AMPA and NMDA r eceptors are clustered at synapses with a density estimated to be on the or der of 10(4) receptors/mu m(2), which corresponds to similar to 400 recepto rs/synapse. Meanwhile there is no reduction in the extrasynaptic receptor d ensity, which indicates that the aggregation of the existing pool of recept ors is not the primary mechanism of glutamate receptor clustering. Furtherm ore our data suggest that the ratio of AMPA to NMDA receptor density may be regulated to be close to one in all dendritic locations. We also demonstra te that synaptic AMPA and NMDA receptor clusters form with a similar time c ourse during synaptogenesis and that functional AMPA receptors cluster inde pendently of activity and glutamate receptor activation, including followin g the deletion of the NMDA receptor NR1 subunit. Thus glutamate receptor ac tivation is not necessary for the insertion, clustering, and activation of functional AMPA receptors during synapse formation, and this process is lik ely controlled by an activity-independent signal.