Rapid recruitment of NMDA receptor transport packets to nascent synapses

Abstract

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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Figure 1: NR1 and GluR1 were present as clusters in dendrites of cortical neurons before synaptogenesis.
Figure 2: Glutamate receptor fusion proteins were correctly targeted to clusters in dendrites of cortical neurons.
Figure 3: NR1 clusters were highly mobile in dendrites of cortical neurons.
Figure 4: GluR1 clusters were less mobile than NR1 clusters.
Figure 5: Molecular and pharmacological characterization of mobile NR1 transport packets.
Figure 6: Rapid recruitment of NMDARs to sites of contact initiated by axon growth cone filopodia.
Figure 7: Recruitment of NMDARs to sites of presynaptic active zones.
Figure 8: Recruitment of GluR2 and PSD-95 to sites of NR1 immobilization.

References

  1. 1

    O'Brien, R.J. et al. The development of excitatory synapses in cultured spinal neurons. J. Neurosci. 17, 7339–7350 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Rao, A., Kim, E., Sheng, M. & Craig, A.M. Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J. Neurosci. 18, 1217–1229 (1998).

    CAS  Article  Google Scholar 

  3. 3

    Kraszewski, K. et al. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J. Neurosci. 15, 4328–4342 (1995).

    CAS  Article  Google Scholar 

  4. 4

    Dai, Z. & Peng, H.B. Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis. Mol. Cell Neurosci. 7, 443–452 (1996).

    CAS  Article  Google Scholar 

  5. 5

    Ahmari, S.E., Buchanan, J. & Smith, S.J. Assembly of presynaptic active zones from cytoplasmic transport packets. Nat. Neurosci. 3, 445–451 (2000).

    CAS  Article  Google Scholar 

  6. 6

    Zhai, R.G. et al. Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29, 131–143 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Ziv, N.E. & Garner, C.C. Principles of glutamatergic synapse formation: seeing the forest for the trees. Curr. Opin. Neurobiol. 11, 536–543 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Okabe, S., Kim, H.D., Miwa, A., Kuriu, T. & Okado, H. Continual remodeling of postsynaptic density and its regulation by synaptic activity. Nat. Neurosci. 2, 804–811 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Friedman, H.V., Bresler, T., Garner, C.C. & Ziv, N.E. Assembly of new individual excitatory synapses: time course and temporal order of synaptic molecule recruitment. Neuron 27, 57–69 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Okabe, S., Miwa, A. & Okado, H. Spine formation and correlated assembly of presynaptic and postsynaptic molecules. J. Neurosci. 21, 6105–6114 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Bresler, T. et al. The dynamics of SAP90/PSD-95 recruitment to new synaptic junctions. Mol. Cell. Neurosci. 18, 149–167 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Marrs, G.S., Green, S.H. & Dailey, M.E. Rapid formation and remodeling of postsynaptic densities in developing dendrites. Nat. Neurosci. 4, 1006–1013 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Prange, O. & Murphy, T.H. Modular transport of postsynaptic density-95 clusters and association with stable spine precursors during early development of cortical neurons. J. Neurosci. 21, 9325–9333 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Durand, G.M., Kovalchuk, Y. & Konnerth, A. Long-term potentiation and functional synapse induction in developing hippocampus. Nature 381, 71–75 (1996).

    CAS  Article  Google Scholar 

  15. 15

    Wu, G., Malinow, R. & Cline, H.T. Maturation of a central glutamatergic synapse. Science 274, 972–976 (1996).

    CAS  Article  Google Scholar 

  16. 16

    Isaac, J.T., Crair, M.C., Nicoll, R.A. & Malenka, R.C. Silent synapses during development of thalamocortical inputs. Neuron 18, 269–280 (1997).

    CAS  Article  Google Scholar 

  17. 17

    Rumpel, S., Hatt, H. & Gottmann, K. Silent synapses in the developing rat visual cortex: evidence for postsynaptic expression of synaptic plasticity. J. Neurosci. 18, 8863–8874 (1998).

    CAS  Article  Google Scholar 

  18. 18

    Liao, D., Zhang, X., O'Brien, R., Ehlers, M.D. & Huganir, R.L. Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons. Nat. Neurosci. 2, 37–43 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Petralia, R.S. et al. Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses. Nat. Neurosci. 2, 31–36 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Cottrell, J.R., Dube, G.R., Egles, C. & Liu, G. Distribution, density, and clustering of functional glutamate receptors before and after synaptogenesis in hippocampal neurons. J. Neurophysiol. 84, 1573–1587 (2000).

    CAS  Article  Google Scholar 

  21. 21

    Scannevin, R.H. & Huganir, R.L. Postsynaptic organization and regulation of excitatory synapses. Nat. Reviews Neurosci. 1, 133–141 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Sheng, M. & Lee, S.H. AMPA receptor trafficking and the control of synaptic transmission. Cell 105, 825–828 (2001).

    CAS  Article  Google Scholar 

  23. 23

    McAllister, A.K. & Stevens, C.F. Nonsaturation of AMPA and NMDA receptors at hippocampal synapses. Proc. Natl. Acad. Sci. USA 97, 6173–6178 (2000).

    CAS  Article  Google Scholar 

  24. 24

    Craig, A.M., Blackstone, C.D., Huganir, R.L. & Banker, G. The distribution of glutamate receptors in cultured rat hippocampal neurons: postsynaptic clustering of AMPA-selective subunits. Neuron 10, 1055–1068 (1993).

    CAS  Article  Google Scholar 

  25. 25

    Mammen, A.L., Huganir, R.L. & O'Brien, R.J. Redistribution and stabilization of cell surface glutamate receptors during synapse formation. J. Neurosci. 17, 7351–7358 (1997).

    CAS  Article  Google Scholar 

  26. 26

    Niethammer, M., Kim, E. & Sheng, M. Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J. Neurosci. 16, 2157–2163 (1996).

    CAS  Article  Google Scholar 

  27. 27

    Arnold, D.B. & Clapham, D.E. Molecular determinants for subcellular localization of PSD-95 with an interacting K+ channel. Neuron 23, 149–157 (1999).

    CAS  Article  Google Scholar 

  28. 28

    Goldstein, L.S. & Yang, Z. Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annu. Rev. Neurosci. 23, 39–71 (2000).

    CAS  Article  Google Scholar 

  29. 29

    Allison, D.W., Chervin, A.S., Gelfand, V.I. & Craig, A.M. Postsynaptic scaffolds of excitatory and inhibitory synapses in hippocampal neurons: maintenance of core components independent of actin filaments and microtubules. J. Neurosci. 20, 4545–4554 (2000).

    CAS  Article  Google Scholar 

  30. 30

    Cooper, M.W. & Smith, S.J. A real-time analysis of growth cone-target cell interactions during the formation of stable contacts between hippocampal neurons in culture. J. Neurobiol. 23, 814–828 (1992).

    CAS  Article  Google Scholar 

  31. 31

    Dailey, M.E. & Smith, S.J. The dynamics of dendritic structure in developing hippocampal slices. J. Neurosci. 16, 2983–2994 (1996).

    CAS  Article  Google Scholar 

  32. 32

    Ziv, N.E. & Smith, S.J. Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17, 91–102 (1996).

    CAS  Article  Google Scholar 

  33. 33

    Ryan, T.A. et al. The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11, 713–724 (1993).

    CAS  Article  Google Scholar 

  34. 34

    Shi, S.H. et al. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284, 1811–1816 (1999).

    CAS  Article  Google Scholar 

  35. 35

    Ehlers, M.D. Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting. Neuron 28, 511–525 (2000).

    CAS  Article  Google Scholar 

  36. 36

    Setou, M., Nakagawa, T., Seog, D.H. & Hirokawa, N. Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport. Science 288, 1796–1802 (2000).

    CAS  Article  Google Scholar 

  37. 37

    Migaud, M. et al. Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396, 433–439 (1998).

    CAS  Article  Google Scholar 

  38. 38

    Chen, L. et al. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943 (2000).

    CAS  Article  Google Scholar 

  39. 39

    Craig, A.M., Blackstone, C.D., Huganir, R.L. & Banker, G. Selective clustering of glutamate and γ-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc. Natl. Acad. Sci. USA 91, 12373–12377 (1994).

    CAS  Article  Google Scholar 

  40. 40

    Verderio, C., Coco, S., Fumagalli, G. & Matteoli, M. Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis. J. Cell. Biol. 126, 1527–1536 (1994).

    CAS  Article  Google Scholar 

  41. 41

    Benson, D.L. & Cohen, P.A. Activity-independent segregation of excitatory and inhibitory synaptic terminals in cultured hippocampal neurons. J. Neurosci. 16, 6424–6432 (1996).

    CAS  Article  Google Scholar 

  42. 42

    Verhage, M. et al. Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287, 864–869 (2000).

    CAS  Article  Google Scholar 

  43. 43

    Schoch, S. et al. SNARE function analyzed in synaptobrevin/VAMP knockout mice. Science 294, 1117–1122 (2001).

    CAS  Article  Google Scholar 

  44. 44

    Washbourne, P. et al. Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat. Neurosci. 5, 19–26 (2002).

    CAS  Article  Google Scholar 

  45. 45

    Benson, D.L. & Tanaka, H. N-cadherin redistribution during synaptogenesis in hippocampal neurons. J. Neurosci. 18, 6892–6904 (1998).

    CAS  Article  Google Scholar 

  46. 46

    Dalva, M.B. et al. EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 103, 945–956 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Scheiffele, P., Fan, J., Choih, J., Fetter, R. & Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 101, 657–669 (2000).

    CAS  Article  Google Scholar 

  48. 48

    Marshall, J., Molloy, R., Moss, G.W., Howe, J.R. & Hughes, T.E. The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron 14, 211–215 (1995).

    CAS  Article  Google Scholar 

  49. 49

    Bekkers, J.M. & Stevens, C.F. NMDA and non-NMDA receptors are co-localized at individual excitatory synapses in cultured rat hippocampus. Nature 341, 230–233 (1989).

    CAS  Article  Google Scholar 

  50. 50

    Aarts, L.H. et al. B-50/GAP-43-induced formation of filopodia depends on Rho-GTPase. Mol. Biol. Cell 9, 1279–1292 (1998).

    CAS  Article  Google Scholar 

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Acknowledgements

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.

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Correspondence to A. Kimberley McAllister.

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Supplementary information

Supplementary Fig. 1.

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)

Supplementary Fig. 2.

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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Washbourne, P., Bennett, J. & McAllister, A. Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nat Neurosci 5, 751–759 (2002). https://doi.org/10.1038/nn883

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