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Data availability statement: The unpublished data used in this study are available from the corresponding author upon reasonable request.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