Skip to main content
SpringerLink
Log in

Live-Cell Superresolution Imaging of Retrograde Axonal Trafficking Using Pulse–Chase Labeling in Cultured Hippocampal Neurons

  • Protocol
  • First Online:
Membrane Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2473))

Abstract

The entanglement of long axons found in cultured dissociated hippocampal neurons restricts the analysis of the machinery underlying directed axonal trafficking. Further, hippocampal neurons exhibit “en passant” presynapses that may confound the analysis of long-range retrograde axonal transport. To solve these issues, we and others have developed microfluid-based methods to specifically follow the fates of the retrograde axonal cargoes following pulse–chase labeling by super-resolution live-cell imaging, and automatically tracking their directed transport and analyzing their kinetical properties. These methods have allowed us to visualize the trafficking of fluorescently tagged signaling endosomes and autophagosomes derived from axonal terminals and resolve their localizations and movements with high spatial and temporal accuracy. In this chapter, we describe how to use a commercially available microfluidic device to enable the labeling and tracking of retrograde axonal carriers, including (1) how to culture and transfect rat hippocampal neurons in the microfluidic device; (2) how to perform pulse–chase to label specific populations of retrograde axonal carriers; and (3) how to conduct the automatic tracking and data analysis using open-source software.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, Jeon NL (2005) A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2(8):599–605. https://doi.org/10.1038/nmeth777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Taylor AM, Jeon NL (2010) Micro-scale and microfluidic devices for neurobiology. Curr Opin Neurobiol 20(5):640–647. https://doi.org/10.1016/j.conb.2010.07.011

    Article  CAS  PubMed  Google Scholar 

  3. Wang J, Ren L, Li L, Liu W, Zhou J, Yu W et al (2009) Microfluidics: a new cosset for neurobiology. Lab Chip 9(5):644–652. https://doi.org/10.1039/b813495b

    Article  CAS  PubMed  Google Scholar 

  4. Spencer RL, Bland ST (2019) Chapter 5—Hippocampus and hippocampal neurons. In: Fink G (ed) Stress: physiology, biochemistry, and pathology. Academic Press, Cambridge, Massachusetts, pp 57–68

    Google Scholar 

  5. Nguyen QT, Son YJ, Sanes JR, Lichtman JW (2000) Nerve terminals form but fail to mature when postsynaptic differentiation is blocked: in vivo analysis using mammalian nerve-muscle chimeras. J Neurosci 20(16):6077–6086. https://doi.org/10.1523/jneurosci.20-16-06077.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Grimm A, Winter N, Rattay TW, Härtig F, Dammeier NM, Auffenberg E et al (2017) A look inside the nerve—morphology of nerve fascicles in healthy controls and patients with polyneuropathy. Clin Neurophysiol 128(12):2521–2526. https://doi.org/10.1016/j.clinph.2017.08.022

    Article  PubMed  Google Scholar 

  7. Wang T, Martin S, Nguyen TH, Harper CB, Gormal RS, Martinez-Marmol R et al (2016) Flux of signalling endosomes undergoing axonal retrograde transport is encoded by presynaptic activity and TrkB. Nat Commun 7:12976. https://doi.org/10.1038/ncomms12976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang T, Martin S, Papadopulos A, Harper CB, Mavlyutov TA, Niranjan D, et al. (2015) Control of autophagosome axonal retrograde flux by presynaptic activity unveiled using botulinum neurotoxin type a. J Neurosci. 35(15):6179–6194.

    Google Scholar 

  9. Restani L, Giribaldi F, Manich M, Bercsenyi K, Menendez G, Rossetto O et al (2012) Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons. PLoS Pathog 8(12):e1003087. https://doi.org/10.1371/journal.ppat.1003087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zweifel LS, Kuruvilla R, Ginty DD (2005) Functions and mechanisms of retrograde neurotrophin signalling. Nat Rev Neurosci 6(8):615–625. https://doi.org/10.1038/nrn1727

    Article  CAS  PubMed  Google Scholar 

  11. Harrington AW, Ginty DD (2013) Long-distance retrograde neurotrophic factor signalling in neurons. Nat Rev Neurosci 14(3):177–187. https://doi.org/10.1038/nrn3253

    Article  CAS  PubMed  Google Scholar 

  12. Maday S, Twelvetrees AE, Moughamian AJ, Holzbaur EL (2014) Axonal transport: cargo-specific mechanisms of motility and regulation. Neuron 84(2):292–309. https://doi.org/10.1016/j.neuron.2014.10.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gowrishankar S, Yuan P, Wu Y, Schrag M, Paradise S, Grutzendler J et al (2015) Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's disease amyloid plaques. Proc Natl Acad Sci U S A 112(28):E3699–E3708. https://doi.org/10.1073/pnas.1510329112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yue Z (2007) Regulation of neuronal autophagy in axon: implication of autophagy in axonal function and dysfunction/degeneration. Autophagy 3(2):139–141. https://doi.org/10.4161/auto.3602

    Article  CAS  PubMed  Google Scholar 

  15. Farfel-Becker T, Roney JC, Cheng X-T, Li S, Cuddy SR, Sheng Z-H (2019) Neuronal Soma-derived degradative lysosomes are continuously delivered to distal axons to maintain local degradation capacity. Cell Rep 28(1):51–64.e4. https://doi.org/10.1016/j.celrep.2019.06.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee S, Sato Y, Nixon RA (2011) Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy. J Neurosci 31(21):7817–7830. https://doi.org/10.1523/jneurosci.6412-10.2011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhou R, Han B, Xia C, Zhuang X (2019) Membrane-associated periodic skeleton is a signaling platform for RTK transactivation in neurons. Science 365(6456):929–934. https://doi.org/10.1126/science.aaw5937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Li H, Yang J, Tian C, Diao M, Wang Q, Zhao S et al (2020) Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging. Nat Commun 11(1):5699. https://doi.org/10.1038/s41467-020-19510-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Joensuu M, Padmanabhan P, Durisic N, Bademosi AT, Cooper-Williams E, Morrow IC et al (2016) Subdiffractional tracking of internalized molecules reveals heterogeneous motion states of synaptic vesicles. J Cell Biol 215(2):277–292. https://doi.org/10.1083/jcb.201604001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bademosi AT, Lauwers E, Padmanabhan P, Odierna L, Chai YJ, Papadopulos A et al (2017) In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters. Nat Commun 8:13660. https://doi.org/10.1038/ncomms13660

    Article  CAS  PubMed  Google Scholar 

  21. Steketee MB, Moysidis SN, Jin XL, Weinstein JE, Pita-Thomas W, Raju HB et al (2011) Nanoparticle-mediated signaling endosome localization regulates growth cone motility and neurite growth. Proc Natl Acad Sci U S A 108(47):19042–19047. https://doi.org/10.1073/pnas.1019624108

    Article  PubMed  PubMed Central  Google Scholar 

  22. Zhou B, Cai Q, Xie Y, Sheng ZH (2012) Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons. Cell Rep 2(1):42–51. https://doi.org/10.1016/j.celrep.2012.06.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Marlin MC, Li G (2015) Biogenesis and function of the NGF/TrkA signaling endosome. Int Rev Cell Mol Biol 314:239–257. https://doi.org/10.1016/bs.ircmb.2014.10.002

    Article  CAS  PubMed  Google Scholar 

  24. Harper CB, Martin S, Nguyen TH, Daniels SJ, Lavidis NA, Popoff MR et al (2011) Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem 286(41):35966–35976. https://doi.org/10.1074/jbc.M111.283879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Harper CB, Papadopulos A, Martin S, Matthews DR, Morgan GP, Nguyen TH et al (2016) Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles. Sci Rep 6:19654. https://doi.org/10.1038/srep19654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Harper CB, Popoff MR, McCluskey A, Robinson PJ, Meunier FA (2013) Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors. Trends Cell Biol 23(2):90–101. https://doi.org/10.1016/j.tcb.2012.10.007

    Article  CAS  PubMed  Google Scholar 

  27. Guedes-Dias P, Holzbaur ELF (2019) Axonal transport: driving synaptic function. Science 366:6462. https://doi.org/10.1126/science.aaw9997

    Article  CAS  Google Scholar 

  28. Reck-Peterson SL, Redwine WB, Vale RD, Carter AP (2018) The cytoplasmic dynein transport machinery and its many cargoes. Nat Rev Mol Cell Biol 19(6):382–398. https://doi.org/10.1038/s41580-018-0004-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li P, Bademosi AT, Luo J, Meunier FA (2018) Actin remodeling in regulated exocytosis: toward a mesoscopic view. Trends Cell Biol 28(9):685–697. https://doi.org/10.1016/j.tcb.2018.04.004

    Article  CAS  PubMed  Google Scholar 

  30. Pan X, Zhou Y, Hotulainen P, Meunier FA, Wang T (2021) The axonal radial contractility: structural basis underlying a new form of neural plasticity. BioEssays 43:e2100033. https://doi.org/10.1002/bies.202100033

    Article  PubMed  Google Scholar 

  31. Wang T, Li W, Martin S, Papadopulos A, Joensuu M, Liu C et al (2020) Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability. J Cell Biol 219(5):e201902001. https://doi.org/10.1083/jcb.201902001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Costa AR, Sousa SC, Pinto-Costa R, Mateus JC, Lopes CD, Costa AC et al (2020) The membrane periodic skeleton is an actomyosin network that regulates axonal diameter and conduction. Elife 9:e55471. https://doi.org/10.7554/eLife.55471

    Article  PubMed  PubMed Central  Google Scholar 

  33. Assaife-Lopes N, Sousa VC, Pereira DB, Ribeiro JA, Sebastião AM (2014) Regulation of TrkB receptor translocation to lipid rafts by adenosine A2A receptors and its functional implications for BDNF-induced regulation of synaptic plasticity. Purinergic Signalling 10(2):251–267. https://doi.org/10.1007/s11302-013-9383-2

    Article  CAS  PubMed  Google Scholar 

  34. Joensuu M, Martínez-Mármol R, Padmanabhan P, Glass NR, Durisic N, Pelekanos M et al (2017) Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules. Nat Protoc 12(12):2590–2622. https://doi.org/10.1038/nprot.2017.116

    Article  CAS  PubMed  Google Scholar 

  35. Maday S, Wallace KE, Holzbaur ELF (2012) Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons. J Cell Biol 196(4):407–417. https://doi.org/10.1083/jcb.201106120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS et al (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793):1642–1645. https://doi.org/10.1126/science.1127344

    Article  PubMed  Google Scholar 

  37. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  38. Chaumont F, Dallongeville S, Olivo-Marin J-C (2011) ICY: A new open-source community image processing software

    Google Scholar 

  39. Maucort G, Kasula R, Papadopulos A, Nieminen TA, Rubinsztein-Dunlop H, Meunier FA (2014) Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells. PLoS One 9(1):e87242. https://doi.org/10.1371/journal.pone.0087242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Couesnon A, Shimizu T, Popoff MR (2009) Differential entry of botulinum neurotoxin a into neuronal and intestinal cells. Cell Microbiol 11(2):289–308. https://doi.org/10.1111/j.1462-5822.2008.01253.x

    Article  CAS  PubMed  Google Scholar 

  41. Olivier N, Keller D, Rajan VS, Gönczy P, Manley S (2013) Simple buffers for 3D STORM microscopy. Biomed Opt Express 4(6):885–899. https://doi.org/10.1364/BOE.4.000885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Szczurek A, Contu F, Hoang A, Dobrucki J, Mai S (2018) Aqueous mounting media increasing tissue translucence improve image quality in structured illumination microscopy of thick biological specimen. Sci Rep 8(1):13971. https://doi.org/10.1038/s41598-018-32191-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Foundation of China (31871036), the Science and Technology Commission of Shanghai Municipality grant 20ZR1436600 and Pujiang Fellowship 20PJ1410300 to TW and the Australian Research Council grant LE130100078, and the National Health and Medical Research Council grants GNT1155794 and GNT1120381 to FAM.

Data availability statement: The unpublished data used in this study are available from the corresponding author upon reasonable request.

Author information

Authors and Affiliations

  1. Center for Brain Science, School of Life Science and Technology, Shanghaitech University, Shanghai, China

    Tong Wang

  2. Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia

    Frédéric A. Meunier

Authors
  1. Tong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frédéric A. Meunier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tong Wang .

Editor information

Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, CO, USA

    Jingshi Shen

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wang, T., Meunier, F.A. (2022). Live-Cell Superresolution Imaging of Retrograde Axonal Trafficking Using Pulse–Chase Labeling in Cultured Hippocampal Neurons. In: Shen, J. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 2473. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2209-4_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2209-4_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2208-7

  • Online ISBN: 978-1-0716-2209-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation