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Design and implementation of in vivo imaging of neural injury responses in the adult Drosophila wing

Nature Protocols volume 8, pages 810–819 (2013)Cite this article

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Abstract

Live-imaging technology has markedly advanced in the field of neural injury and axon degeneration; however, studies are still predominantly performed in in vitro settings such as cultured neuronal cells or in model organisms such as Caenorhabditis elegans in which axons lack glial wrappings. We recently developed a new in vivo model for adult-stage neural injury in Drosophila melanogaster, using the highly accessible wing of the animal. Because the Drosophila wing is translucent and dispensable for survival, it allows clear and direct visualization of injury-induced progressive responses of axons and glia highlighted by fluorescent protein (FP) markers in live animals over time. Moreover, unlike previous Drosophila models of neural injury, this procedure does not require dissection of the CNS. Thus, the key preparation steps for in vivo imaging of the neural injury response described in this protocol can be completed within 30 min.

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Figure 1: The neuronal and glial components of the Drosophila wing.
Figure 2: An overview of the experimental procedure.
Figure 3: The filter settings for visualization of GFP and mCherry fluorescence of the wing nerve.
Figure 4: The wing cut preparation.
Figure 5: The preparation for live-imaging of the wing nerve.
Figure 6: The preparation for quick imaging and scoring of the nerve.
Figure 7: Representative images of neural injury–induced axonal and glial responses.

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Acknowledgements

We are grateful to M. Landgraf, B. White, T. Lee and the Bloomington Drosophila Stock Center at Indiana University for providing transgenic flies used in this work, as well as to G. Rubin, B. Pfeiffer and N. Aijoscha for sharing unpublished reagents. We also thank X. Teng and M. Geary for their excellent technical assistance, M. Parisi for the nsyb-LexA::GAD>LexAop-rCD2-GFP recombinant fly line, and L.-Y. Hao, S.-Y. Shieh and the other members of the Bonini lab for their thoughtful discussions. N.M.B. is an investigator of the Howard Hughes Medical Institute. This work was supported by the National Institutes of Health EUREKA award (grant no. 1R01NS066312) and an Ellison Medical Foundation Senior Scholar in Aging Award to N.M.B.

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Authors and Affiliations

  1. Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Yanshan Fang, Lorena Soares & Nancy M Bonini

  2. Howard Hughes Medical Institute, Philadelphia, Pennsylvania, USA

    Yanshan Fang & Nancy M Bonini

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  1. Yanshan Fang
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  2. Lorena Soares
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  3. Nancy M Bonini
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Contributions

Y.F., L.S. and N.M.B. conceived the project and developed the protocol; Y.F. designed and performed the experiments and analyzed the results; Y.F. and N.M.B. wrote the paper.

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Correspondence to Nancy M Bonini.

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Fang, Y., Soares, L. & Bonini, N. Design and implementation of in vivo imaging of neural injury responses in the adult Drosophila wing. Nat Protoc 8, 810–819 (2013). https://doi.org/10.1038/nprot.2013.042

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