Although many of the molecules involved in synaptogenesis have been identified, the sequence and kinetics of synapse assembly in the central nervous system (CNS) remain largely unknown. We used simultaneous time-lapse imaging of fluorescent glutamate receptor subunits and presynaptic proteins in rat cortical neurons in vitro to determine the dynamics and time course of N-methyl-D-aspartate receptor (NMDAR) recruitment to nascent synapses. We found that both NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits are present in mobile transport packets in neurons before and during synaptogenesis. NMDAR transport packets are more mobile than AMPAR subunits, moving along microtubules at about 4 μm/min, and are recruited to sites of axodendritic contact within minutes. Whereas NMDAR recruitment to new synapses can be either concurrent with or independent of the protein PSD-95, AMPARs are recruited with a slower time course. Thus, glutamatergic synapses can form rapidly by the sequential delivery of modular transport packets containing glutamate receptors.
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We thank J. Sullivan for help in constructing the NR1 and GluR1 fusion constructs; S. Heinemann for providing NR1 and GluR1 cDNAs; M. Sheng for providing PSD-95-EGFP27; R. Scheller for VAMP2-EGFP5; N. Perrone-Bizzozero for GAP43-EGFP50; S. Vicini, J. Luo and Z. Fu for the EGFP-NR1 (N-terminal fusion construct); R. Huganir for his gift of guinea pig anti-GluR1 antibody and the Jones lab at UC Davis for sharing resources. Thanks also to the Ehlers lab at Duke University for the lipofection protocol and to K. Murray, S. Sabo and W.M. Usrey for reading the manuscript. This work was supported by the Alfred P. Sloan Foundation, the Pew Charitable Trusts, the March of Dimes and NIH RO1 EY13584 (A.K.M.). P.W. is a M.I.N.D. Institute Scholar.
The authors declare no competing financial interests.
Movement of an NMDAR transport packet. A 3 d.i.v. cerebral cortical neuron transfected with NR1-DsRed and imaged at 35°C, 24 h after transfection. This time-lapse movie demonstrates movement of an NMDAR transport packet (arrow) in a retrograde direction along a proximal dendrite. NR1-DsRed is in red. Images were acquired every 2 min. A total of 17 frames, corresponding to 32 min of imaging, is compressed to 2 s. (AVI 1553 kb)
Recruitment of NR1-DsRed to a site of contact with an axon growth cone filopodium. Cortical neurons 3 d.i.v. 'trans' cotransfected with NR1-DsRed (red) and GAP43-EGFP (green) were imaged at 34°C, 16 h after transfection. Growth cone filopodia contacted the NR1-positive dendrite repeatedly, causing recruitment of an NMDAR transport packet to the site of contact. Both the contact and the NMDAR transport packet were then stabilized. Images were acquired every 90 s. A total of 15 frames, corresponding to 21 min of imaging, is compressed to 2 s. (AVI 1321 kb)
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