Abstract
Live-imaging of axonal cargoes within central nervous system has been a long-lasting interest for neurobiologists as axonal transport plays critical roles in neuronal growth, function, and survival. Many kinds of cargoes are transported within axons, including synaptic vesicles and a variety of membrane-bound and membrane-less organelles. Imaging these cargoes at high spatial and temporal resolution, and within living brains, is technically very challenging. Here, we describe a quantitative method, based on customized mounting chambers, allowing live-imaging of axonal cargoes transported within the maturing brain of the fruit fly, Drosophila melanogaster. With this method, we could visualize in real time, using confocal microscopy, cargoes transported along axons. Our protocol is simple and easy to set up, as brains are mounted in our imaging chambers and ready to be imaged in about 1 h. Another advantage of our method is that it can be combined with pharmacological treatments or super-resolution microscopy.
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Acknowledgments
We thank I. Gaspar for his help in image acquisition and analysis. We are also grateful to L. Burger from the EMBL mechanical workshop for machining the metal rings and G. Durand for the technical drawing of these rings. We thank also Y. Belyaev and S. Terjung from the EMBL microscopy platform (AMLF) for their advice in selecting the appropriate microscope for imaging. We thank W. Huebner for his daily help at EMBL, and F. Brau from the IPMC imaging facility. C.M. was supported by short-term fellowships from EMBO, FEBS and P3 (http://www.p-cube.eu/), and by a long-term fellowship from “Ville de Nice.” Development of this protocol was supported by grants (ATIP/CNRS, FRM Implantation Nouvelles Equipes, ARC Fixe, HFSP Career Development Award and ANR JCJC) to F.B.
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Live-imaging of membranous vesicles transported within a bundle of axons (mushroom body gamma neurons). The fluorescent reporter myr-tdTomato was expressed specifically in the mushroom body gamma neurons. Both axonal plasma membranes and bidirectional motile membrane vesicles are labeled. The size of the imaged region is 380 × 250 pixels (53.14 × 34.96 μm). Scale bar: 5 μm. Acquisition rate: 1 frame/s. (Adapted from [17]). (MP4 14933 kb)
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Medioni, C., Ephrussi, A., Besse, F. (2022). Live-Imaging of Axonal Cargoes in Drosophila Brain Explants Using Confocal Microscopy. In: Dahlmanns, J., Dahlmanns, M. (eds) Synaptic Vesicles. Methods in Molecular Biology, vol 2417. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1916-2_2
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DOI: https://doi.org/10.1007/978-1-0716-1916-2_2
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