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A simple method for imaging axonal transport in aging neurons using the adult Drosophila wing

Nature Protocols volume 11, pages 1711–1723 (2016)Cite this article

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Abstract

There is growing interest in the link between axonal cargo transport and age-associated neuronal dysfunction. The study of axonal transport in neurons of adult animals requires intravital or ex vivo imaging approaches, which are laborious and expensive in vertebrate models. We describe simple, noninvasive procedures for imaging cargo motility within axons using sensory neurons of the translucent Drosophila wing. A key aspect is a method for mounting the intact fly that allows detailed imaging of transport in wing neurons. Coupled with existing genetic tools in Drosophila, this is a tractable system for studying axonal transport over the life span of an animal and thus for characterization of the relationship between cargo dynamics, neuronal aging and disease. Preparation of a sample for imaging takes ∼5 min, with transport typically filmed for 2–3 min per wing. We also document procedures for the quantification of transport parameters from the acquired images and describe how the protocol can be adapted to study other cell biological processes in aging neurons.

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Figure 1: Imaging organelle transport in the nervous system of the adult Drosophila wing.
Figure 2: Overview of the experimental procedure.
Figure 3: Key steps in the preparation of the imaging chamber.
Figure 4: Visualization of mitochondrial fission and fusion events in wing neurons.
Figure 5: Representative results with different combinations of drivers and cargo markers in wing neurons.

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Acknowledgements

We are grateful to the LMB Media Kitchen for fly food preparation, N. Grant from LMB Visual Aids for help with Figure 3 and Supplementary Video 1, M. Pasche (MRC-LMB) for assistance with the SIM experiments, and D. Brunner (University of Zurich, Switzerland), G. Jefferis (MRC-LMB, UK), A. Whitworth (MRC Mitochondrial Biology Unit, UK) and the Bloomington Drosophila Stock Center (Indiana University) for sharing fly stocks. The development of this protocol was supported by funding to S.L.B. from the UK Medical Research Council (MRC file reference number MC_U105178790). A.V. is the recipient of an NC3Rs David Sainsbury Fellowship (NC/N001753/1).

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Authors and Affiliations

  1. Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, UK

    Alessio Vagnoni & Simon L Bullock

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  1. Alessio Vagnoni
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Contributions

A.V. and S.L.B. conceived the study. A.V. and S.L.B. designed the experiments. A.V. performed the experiments and collected the data. A.V. and S.L.B. wrote the manuscript.

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Correspondence to Alessio Vagnoni or Simon L Bullock.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Assessment of the effects of CO2 exposure on cargo transport.

Quantification of the number of total mitochondria that are motile in dpr+ wing neurons in a 3 min time window when flies are exposed to CO2 for 5 min followed by mounting and immediate imaging (CO2 – long) or exposed to CO2 for 20–30 s followed by mounting and a ~ 5 min recovery period before imaging (CO2 – short). Bar shows mean ± standard error of the mean, with values for each movie shown as magenta circles. For each parameter, there was no statistically significant difference between the two conditions.

Supplementary Figure 2 Structured illumination microscopy (SIM) of L1 vein neurons.

Representative SIM image of the anterior wing margin showing cell bodies and processes of neurons in the L1 vein. Wings were mounted using the procedures described in this article. The genotype of the fly is Appl-Gal4>mCD8::GFP, RedStinger::NLS, which leads to cell membranes and nuclei marked in green and red, respectively. Arrowheads, dendrites; arrows, bundled axons. Note the improved detail in the SIM image compared to the image taken by spinning disk microscopy of the same genotype (Fig. 1b). The image is a projection of a z-stack taken on a Zeiss Elyra imaging system using a 63 1.4 NA PlanApo oil-immersion objective. Scale bar: 5 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 (PDF 238 kb)

Movie capturing the procedure used to mount a fly in the imaging chamber (Steps 8–19), with the chamber partially assembled in advance).

Note that the movie does not show the process of inspecting wings for damage or the whole anesthetization procedure. (MOV 24868 kb)

Representative time-lapse movie of mitochondrial dynamics in axons of dpr+ neurons in the wing arch (L1 vein) at 1 day after eclosion.

The cell body is to the left and the thorax to the right in this and other movies. Note that, as in many other neuronal cell types, a significant fraction of mitochondria are stationary at any one time point. Genotype: dpr-Gal4 UAS-mito::GFP. Width: 50 μm. Movie represents 3 min of real time. (MOV 1154 kb)

Representative time-lapse movie of mitochondrial dynamics in axons of nSyb+ neurons in the L3 vein at 2 days after eclosion.

Genotype: nSyb-lexAlexAop- mCherry::mito. Width: 50 μm. Movie represents 3 min of real-time. (MOV 1177 kb)

Time-lapse movie showing an example of mitochondrial fission in axons of dpr+ neurons in the wing arch.

A stationary mitochondrion (red arrow) undergoes fission to produce a new mitochondrion (yellow arrow) that is motile. Genotype: dpr-Gal4 UAS- mito::GFP. Width: 17 μm. Movie represents 68 s of real time. (MOV 59 kb)

Time-lapse movie showing an example of mitochondrial fusion in axons of dpr+ neurons in the wing arch.

A motile mitochondrion (red arrow) undergoes fusion with a stationary mitochondrion (yellow arrow). The orange arrow marks the movement of the new elongated mitochondrion produced by the fusion event. Genotype: dpr-Gal4 UAS- mito: GFP. Width: 50 μm. Movie represents 266 s of real time. (MOV 275 kb)

Representative time-lapse movie of ANF::EMD dynamics in axons of dpr+ neurons in the wing arch at 2 days after eclosion.

The ANF: EMD signal on dense-core vesicles is masked by cytoplasmic GFP encoded by a UAS-GFP transgene on the dpr-Gal4 chromosome. Genotype: dpr-Gal4 UAS-ANF: EMD. Width: 50 μm. Movie represents 1 min of real time. (MOV 191 kb)

Representative time-lapse movie of ANF::EMD dynamics in axons of Appl+ neurons in the wing arch at 2 days after eclosion.

This genotype allows dense-core vesicles to be visualized clearly. Genotype: Appl-Gal4 UAS-ANF: GFP. Width: 50 μm. Movie represents 2 min of real time. (MOV 821 kb)

Representative time-lapse movie of Rab4: RFP dynamics in axons of dpr+ neurons in the wing arch at 2 days after eclosion.

Genotype: dpr-Gal4UAS-Rab4: RFP. Width: 50 μm. Movie represents 1 min of real time. (MOV 543 kb)

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Vagnoni, A., Bullock, S. A simple method for imaging axonal transport in aging neurons using the adult Drosophila wing. Nat Protoc 11, 1711–1723 (2016). https://doi.org/10.1038/nprot.2016.112

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