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Imaging Axonal Transport in Ex Vivo Central and Peripheral Nerves

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Axonal Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2431))

Abstract

Neurones are highly polarized cells with extensive axonal projections that rely on transport of proteins, RNAs, and organelles in a bidirectional manner to remain healthy. This process, known as axonal transport, can be imaged in real time through epifluorescent imaging of fluorescently labeled proteins, organelles, and other cargoes. While this is most conveniently done in primary neuronal cultures, it is more physiologically relevant when carried out in the context of a developed nerve containing both axons and glia. Here we outline how to image axonal transport ex vivo in sciatic and optic nerves, and the fimbria of the fornix. These methods could be altered to image other fluorescently labeled molecules, as well as different mechanisms of intracellular transport.

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References

  1. Sajic M, Mastrolia V, Lee CY, Trigo D, Sadeghian M, Mosley AJ et al (2013) Impulse conduction increases mitochondrial transport in adult mammalian peripheral nerves in vivo. PLoS Biol. 11(12):e1001754. https://doi.org/10.1371/journal.pbio.1001754

    Article  PubMed  PubMed Central  Google Scholar 

  2. Milde S, Fox AN, Freeman MR, Coleman MP (2013) Deletions within its subcellular targeting domain enhance the axon protective capacity of Nmnat2 in vivo. Sci Rep 3:2567–2567

    Article  Google Scholar 

  3. Misgeld T, Kerschensteiner M, Bareyre FM, Burgess RW, Lichtman JW (2007) Imaging axonal transport of mitochondria in vivo. Nat Methods 4(7):559–561. https://doi.org/10.1038/nmeth1055

    Article  CAS  PubMed  Google Scholar 

  4. Sorbara CD, Wagner NE, Ladwig A, Nikić I, Merkler D, Kleele T et al (2014) Pervasive axonal transport deficits in multiple sclerosis models. Neuron. 84(6):1183–1190. https://doi.org/10.1016/j.neuron.2014.11.006

    Article  CAS  PubMed  Google Scholar 

  5. Gibbs KL, Kalmar B, Sleigh JN, Greensmith L, Schiavo G (2016) In vivo imaging of axonal transport in murine motor and sensory neurons. J Neurosci Methods 257:26–33. https://doi.org/10.1016/j.jneumeth.2015.09.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bilsland LG, Sahai E, Kelly G, Golding M, Greensmith L, Schiavo G (2010) Deficits in axonal transport precede ALS symptoms in vivo. PNAS 107(47):20523–20528

    Article  CAS  Google Scholar 

  7. Sleigh JN, Tosolini AP, Gordon D, Devoy A, Fratta P, Fisher EMC et al (2020) Mice carrying ALS mutant TDP-43, but not mutant FUS, display in vivo defects in axonal transport of signaling endosomes. Cell Reports 30(11):3655–3662.e2. https://doi.org/10.1016/j.celrep.2020.02.078

    Article  CAS  PubMed  Google Scholar 

  8. Kalinski AL, Kar AN, Craver J, Tosolini AP, Sleigh JN, Lee SJ et al (2019) Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition. J Cell Biol 218(6):1871–1890. https://doi.org/10.1083/jcb.201702187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sleigh JN, Tosolini AP, Schiavo G (2020. [cited 2021 Jan 28]. p. 271–92. (Methods in Molecular Biology). Available from:) In vivo imaging of anterograde and retrograde axonal transport in rodent peripheral nerves. In: Babetto E (ed) Axon degeneration: methods and protocols [Internet]. Springer, New York, NY. https://doi.org/10.1007/978-1-0716-0585-1_20

    Chapter  Google Scholar 

  10. Gould S (2021) Exploring SARM1 as a target to delay programmed axon degeneration [Internet] [Thesis]. University of Cambridge. [cited 2021 Jan 27]. Available from: https://www.repository.cam.ac.uk/handle/1810/316071

    Google Scholar 

  11. Milde S, Adalbert R, Elaman MH, Coleman MP (2015) Axonal transport declines with age in two distinct phases separated by a period of relative stability. Neurobiol Aging 36(2):971–981. https://doi.org/10.1016/j.neurobiolaging.2014.09.018

    Article  PubMed  PubMed Central  Google Scholar 

  12. Adalbert R, Milde S, Durrant C, Ando K, Stygelbout V, Yilmaz Z et al (2018) Interaction between a MAPT variant causing frontotemporal dementia and mutant APP affects axonal transport. Neurobiol Aging 68:68–75. https://doi.org/10.1016/j.neurobiolaging.2018.03.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gilley J, Seereeram A, Ando K, Mosely S, Andrews S, Kerschensteiner M et al (2012) Age-dependent axonal transport and locomotor changes and tau hypophosphorylation in a “P301L” tau knockin mouse. Neurobiol Aging 33(3):621.e1–621.e15. https://doi.org/10.1016/j.neurobiolaging.2011.02.014

    Article  CAS  Google Scholar 

  14. Mandal A, Drerup CM (2019. [cited 2021 Jan 29];13. Available from: https://www.frontiersin.org/articles/10.3389/fncel.2019.00373/full#B135) Axonal transport and mitochondrial function in neurons. Front Cell Neurosci. https://doi.org/10.3389/fncel.2019.00373

  15. Smith GM, Gallo G (2018) The role of mitochondria in axon development and regeneration. Dev Neurobiol 78(3):221–237. https://doi.org/10.1002/dneu.22546

    Article  CAS  PubMed  Google Scholar 

  16. Zhang CL, Ho PL, Kintner DB, Sun D, Chiu SY (2010) Activity-dependent regulation of mitochondrial motility by calcium and Na/K-ATPase at nodes of ranvier of myelinated nerves. J Neurosci 30(10):3555–3566. https://doi.org/10.1523/JNEUROSCI.4551-09.2010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Saotome M, Safiulina D, Szabadkai G, Das S, Fransson Å, Aspenstrom P et al (2008) Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase. PNAS. 105(52):20728–20733

    Article  CAS  Google Scholar 

  18. Andrews S, Gilley J, Coleman MP (2010) Difference tracker: ImageJ plugins for fully automated analysis of multiple axonal transport parameters. J Neurosci Methods 193(2):281–287. https://doi.org/10.1016/j.jneumeth.2010.09.007

    Article  PubMed  Google Scholar 

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Correspondence to Michael Coleman .

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1 Electronic Supplementary Materials

Video 1

Example video generated by imaging ex vivo axonal transport of Venus-labeled NMNAT2 in sciatic nerve. (MP4 19322 kb)

Video 2

Example video of a “straightened” axon video generated by selection of an axon in the field of view of the original video (Video 1). (MP4 711 kb)

Video 3

Example video of tracked particles video generated from a straightened video (Video 2). (MP4 1012 kb)

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

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Gould, S.A., Adalbert, R., Milde, S., Coleman, M. (2022). Imaging Axonal Transport in Ex Vivo Central and Peripheral Nerves. In: Vagnoni, A. (eds) Axonal Transport. Methods in Molecular Biology, vol 2431. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1990-2_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1990-2_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1989-6

  • Online ISBN: 978-1-0716-1990-2

  • eBook Packages: Springer Protocols

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