Abstract
Axonal transport, which is composed of microtubules, motor proteins and a variety of types of cargo, is a prominent feature of neurons. Monitoring these molecular dynamics is important to understand the biological processes of neurons as well as neurodegenerative disorders that are associated with axonal dysfunction. Here, we describe a protocol for monitoring the axonal transport of motor neurons in Drosophila larvae using inverted fluorescence microscopy.
Keywords: Axonal transport, Live imaging, Drosophila, Inverted fluorescence microscopy
Background
Axons are a unique neuronal structure, by which neurons transmit electric and chemical signals to neighboring neurons, muscles and other tissues. Axonal function is maintained by the circular flow or shuttling of vesicles, organelles and materials (Liu et al., 2012; Wong et al., 2012; Alami et al., 2014). Axonal dysfunction is thought to be an early sign of neurodegeneration and a cause of neurodegenerative diseases such as Alzheimer’s, Parkinson’s and motor neuron diseases in humans (Hirokawa et al., 2010; Millecamps and Julien, 2013). However, it is difficult to monitor the process of axonal degeneration in these neurodegenerative diseases in both humans and mammalian models. The Drosophila model is a powerful tool for studying neurodegenerative diseases at the molecular-genetic level and has provided important evidence for therapeutic approaches through the live imaging of neurons in vivo (Shiba-Fukushima et al., 2014; Hosaka et al., 2017). Upright fluorescence microscopy techniques are generally used to analyze the live imaging of tissues and organ cultures. However, inverted fluorescence microscopy has seen an increase in demand due to its broad utility and extensibility. Here, we introduce our protocol to analyze the axonal transport of motor neurons in the third-instar larvae of Drosophila using inverted fluorescence microscopy.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Recipes
Acknowledgments
This study was funded by the Grant-in-Aid for Scientific Research (C, 16K09679 [T.I.]; B, 17H04049 [Y.I.]) from MEXT in Japan; additionally, partial support was obtained by a grant from Otsuka Pharmaceuticals (N.H. and Y.I.). This protocol is adapted from Hosaka et al., 2017. The authors declare no conflict of interest.
References
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