Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Live-cell imaging of receptors around postsynaptic membranes

Abstract

This protocol describes how to image the trafficking of glutamate receptors around excitatory postsynaptic membrane formed on an adhesion protein–coated glass surface. The protocol was developed to clarify how receptors move during the induction of synaptic plasticity. Dissociated neurons are cultured on a coverslip coated with neurexin, which induces the formation of postsynaptic membrane-like structures on the glass surface. A glutamate receptor tagged with a fluorescent protein is then transfected into neurons, and it is observed with total internal reflection fluorescence microscopy. The whole process takes about 3 weeks. Changes in the amount of cell-surface receptors caused by neuronal activities can be quantified, and individual exocytosis events of receptors can be clearly observed around the pseudo-postsynaptic membrane. This protocol has potential applications for studies of movements of membrane proteins around other specialized regions of the cell membrane, such as the inhibitory postsynaptic membrane, the presynaptic membrane or the immunological synapses.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Visual depiction of the protocol.
Figure 2: Coating a glass coverslip with NRX.
Figure 3: Outline of the NRX purification procedure (Steps 1–9).
Figure 4: PSLM and normal synapses observed with TIRFM or with conventional epifluorescence.
Figure 5: Formation of PSLM on NRX-coated glass with or without expression of NLG, or on NRX- or non-NRX-coated glass with NLG.
Figure 6: The experimental chamber holding a glass coverslip on which neurons have been cultured.
Figure 7: Recording setup for live-cell imaging.
Figure 8: Examples of live-cell imaging experiments.

Similar content being viewed by others

References

  1. Okabe, S. Molecular anatomy of the postsynaptic density. Mol. Cell Neurosci. 34, 503–518 (2007).

    Article  CAS  Google Scholar 

  2. Sheng, M. & Hoogenraad, C.C. The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu. Rev. Biochem. 76, 823–847 (2007).

    Article  CAS  Google Scholar 

  3. Malinow, R. & Malenka, R.C. AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25, 103–126 (2002).

    Article  CAS  Google Scholar 

  4. Shepherd, J.D. & Huganir, R.L. The cell biology of synaptic plasticity: AMPA receptor trafficking. Annu. Rev. Cell Dev. Biol. 23, 613–643 (2007).

    Article  CAS  Google Scholar 

  5. Kessels, H.W. & Malinow, R. Synaptic AMPA receptor plasticity and behavior. Neuron 61, 340–350 (2009).

    Article  CAS  Google Scholar 

  6. Hirano, T. Long-term depression and other synaptic plasticity in the cerebellum. Proc. Jpn. Acad. Ser. B 89, 183–195 (2013).

    Article  CAS  Google Scholar 

  7. Klyubin, I., Cullen, W.K., Hu, N.W. & Rowan, M.J. Alzheimer's disease Aβ assemblies mediating rapid disruption of synaptic plasticity and memory. Mol. Brain 5, 25 (2012).

    Article  CAS  Google Scholar 

  8. Lüscher, C. & Malenka, R.C. Drug-evoked synaptic plasticity in addiction: from molecular changes to circuit remodeling. Neuron 69, 650–663 (2011).

    Article  Google Scholar 

  9. Borgdorff, A.J. & Choquet, D. Regulation of AMPA receptor lateral movements. Nature 417, 649–653 (2002).

    Article  CAS  Google Scholar 

  10. Bannai, H., Lévi, S., Schweizer, C., Dahan, M. & Triller, A. Imaging the lateral diffusion of membrane molecules with quantum dots. Nat. Protoc. 1, 2628–2634 (2006).

    Article  CAS  Google Scholar 

  11. Ehlers, M.D., Heine, M., Groc, L., Lee, M.C. & Choquet, D. Diffusional trapping of GluR1 AMPA receptors by input-specific synaptic activity. Neuron 54, 447–460 (2007).

    Article  CAS  Google Scholar 

  12. Yudowski, G.A. et al. Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors. J. Neurosci. 27, 11112–11121 (2007).

    Article  CAS  Google Scholar 

  13. Lin, D.T. et al. Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation. Nat. Neurosci. 12, 879–887 (2009).

    Article  CAS  Google Scholar 

  14. Kennedy, M.J., Davison, I.G., Robinson, C.G. & Ehlers, M.D. Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines. Cell 141, 524–535 (2010).

    Article  CAS  Google Scholar 

  15. Patterson, M.A., Szatmari, E.M. & Yasuda, R. AMPA receptors are exocytosed in stimulated spines and adjacent dendrites in a Ras-ERK–dependent manner during long-term potentiation. Proc. Natl. Acad. Sci. USA 107, 15951–15956 (2010).

    Article  CAS  Google Scholar 

  16. Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001).

    Article  CAS  Google Scholar 

  17. Toomre, D. & Manstein, D.J. Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11, 298–303 (2001).

    Article  CAS  Google Scholar 

  18. Tanaka, H. & Hirano, T. Visualization of subunit-specific delivery of glutamate receptors to postsynaptic membrane during hippocampal long-term potentiation. Cell Rep. 1, 291–298 (2012).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. Graf, E.R., Zhang, X., Jin, S.X., Linhoff, M.W. & Craig, A.M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119, 1013–1026 (2004).

    Article  CAS  Google Scholar 

  21. Südhof, T.C. Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455, 903–911 (2008).

    Article  Google Scholar 

  22. Taguchi, H., Ueno, T., Tadakuma, H., Yoshida, M. & Funatsu, T. Single-molecule observation of protein-protein interactions in the chaperonin system. Nat. Biotechnol. 19, 861–865 (2001).

    Article  CAS  Google Scholar 

  23. Pautot, S., Lee, H., Isacoff, E.Y. & Groves, J.T. Neuronal synapse interaction reconstituted between live cells and supported lipid bilayers. Nat. Chem. Biol. 1, 283–289 (2005).

    Article  CAS  Google Scholar 

  24. Jain, A., Liu, R., Xiang, Y.K. & Ha, T. Single-molecule pull-down for studying protein interactions. Nat. Protoc. 7, 445–452 (2012).

    Article  CAS  Google Scholar 

  25. Miesenböck, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  Google Scholar 

  26. Brewer, G.J., Torricelli, J.R., Evege, E.K. & Price, P.J. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567–576 (1993).

    Article  CAS  Google Scholar 

  27. Thompson, N.L. & Steele, B.L. Total internal reflection with fluorescence correlation spectroscopy. Nat. Protoc. 2, 878–890 (2007).

    Article  CAS  Google Scholar 

  28. Mutch, S.A. et al. Determining the number of specific proteins in cellular compartments by quantitative microscopy. Nat. Protoc. 6, 1953–1968 (2011).

    Article  CAS  Google Scholar 

  29. Makino, H. & Malinow, R. AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis. Neuron 64, 381–390 (2009).

    Article  CAS  Google Scholar 

  30. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  Google Scholar 

  31. Hell, S.W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  Google Scholar 

  32. Willig, K.I., Rizzoli, S.O., Westphal, V., Jahn, R. & Hell, S.W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

    Article  CAS  Google Scholar 

  33. Gustafsson, M.G.L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Article  CAS  Google Scholar 

  34. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  35. Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).

    Article  CAS  Google Scholar 

  36. Van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 991–1009 (2011).

    Article  CAS  Google Scholar 

  37. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  38. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  39. Gould, T.J., Verkhusha, V.V. & Hess, S.T. Imaging biological structures with fluorescence photoactivation localization microscopy. Nat. Protoc. 4, 291–308 (2009).

    Article  CAS  Google Scholar 

  40. Schermelleh, L., Heintzmann, R. & Leonhardt, H. A guide to super-resolution fluorescence microscopy. J. Cell Biol. 190, 165–175 (2010).

    Article  CAS  Google Scholar 

  41. Maglione, M. & Sigrist, S.J. Seeing the forest tree by tree: super-resolution light microscopy meets the neuroscience. Nat. Neurosci. 16, 790–797 (2013).

    Article  CAS  Google Scholar 

  42. Kang, Y., Zhang, X., Dobie, F., Wu, H. & Craig, A.M. Induction of GABAergic postsynaptic differentiation by α-neurexins. J. Biol. Chem. 283, 2323–2334 (2008).

    Article  CAS  Google Scholar 

  43. Midorikawa, M., Tsukamoto, Y., Berglund, K., Ishii, M. & Tachibana, M. Different roles of ribbon-associated and ribbon-free active zones in retinal bipolar cells. Nat. Neurosci. 10, 1268–1276 (2007).

    Article  CAS  Google Scholar 

  44. Mace, E.M. & Orange, J.S. New views of the human NK cell immunological synapse: recent advances enabled by super- and high-resolution imaging techniques. Front. Immunol. 3, 421 (2013).

    Article  Google Scholar 

  45. Thomason, H.A., Scothern, A., McHarg, S. & Garrod, D.R. Desmosomes: adhesive strength and signalling in health and disease. Biochem. J. 429, 419–433 (2010).

    Article  CAS  Google Scholar 

  46. Ivanov, A.I. & Naydenov, N.G. Dynamics and regulation of epithelial adherens junctions: recent discoveries and controversies. Int. Rev. Cell Mol. Biol. 303, 27–99 (2013).

    Article  CAS  Google Scholar 

  47. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

    Article  CAS  Google Scholar 

  48. Kawaguchi, S.Y. & Hirano, T. Integrin α3β1 suppresses long-term potentiation at inhibitory synapses on the cerebellar Purkinje neuron. Mol. Cell Neurosci. 31, 416–426 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Ehrlich, I. & Malinow, R. Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. J. Neurosci. 24, 916–927 (2004).

    Article  CAS  Google Scholar 

  51. Sugiyma, Y., Kawabata, I., Sobue, K. & Okabe, S. Determination of absolute protein numbers in single synapses by a GFP-based calibration technique. Nat. Methods 2, 677–684 (2005).

    Article  Google Scholar 

  52. Varoqueaux, F. et al. Neuroligins determine synapse maturation and function. Neuron 51, 741–754 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Kawaguchi, Y. Tagawa and E. Nakajima for comments on the manuscript. We thank S. Okabe (Tokyo University) for the PSD95-EGFP expression vector. This research was supported by grants-in-aid for scientific research, Global Centers of Excellence program A06 of Kyoto University; Grants for Excellent Graduate Schools from the Ministry of Education, Culture, Sports, Science and Technology; and from the Takeda Science Foundation in Japan.

Author information

Authors and Affiliations

Authors

Contributions

H.T. and T.H. designed the study and wrote the paper. H.T. and S.F. improved the protocol and performed the experiments.

Corresponding author

Correspondence to Tomoo Hirano.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 NRX-coating check.

(a-c) Observation of coated NRX (green) and NLG-HA (magenta) expressed in HEK cells by immunostaining. (b) When NRX is immunostained without permeabilization of the membrane, NRX signal is not detected under a HEK cell (arrowhead). (c) After permeabilization, NRX under a HEK cell is stained, suggesting that tight coupling of NRX and NLG prevents access of the antibody to NRX under a cell. (b-d) NLG-HA-expressing HEK cells often show fan-shaped lamellipodia-like swelling (b,c, arrows) and / or well-extended filopodia (d, arrows). These morphological changes of HEK cells are signs of strong interaction between NRX on the glass surface and NLG in HEK cells. Scale bars, 100 ÎĽm (a), 20 ÎĽm (c).

Supplementary information

Supplementary Figure 1

NRX-coating check. (PDF 119 kb)

High-frequency imaging of GluA1-SEP.

GluA1-SEP (green) exocytosis in the periphery of PSD95-RFP (magenta)-positive area shown in Figure 8b is recorded. The movie starts at 48.8 s after the start of electrical stimulation, and runs in real time. (MOV 242 kb)

Dual-color imaging of GluA1-SEP and PSD95-RFP.

GluA1-SEP (green) exocytosis in the periphery of PSD95-RFP (magenta)-positive area shown in Figure 8d is recorded. The movie starts at 84.6 s after the start of electrical stimulation, and runs in real time. (MOV 1095 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tanaka, H., Fujii, S. & Hirano, T. Live-cell imaging of receptors around postsynaptic membranes. Nat Protoc 9, 76–89 (2014). https://doi.org/10.1038/nprot.2013.171

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.171

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing