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Use of Microfluidics Chambers to Image Axonal transport in Adult Sensory Neurons

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Axonal Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2431))

Abstract

Transport of cargoes along axons is crucial for ensuring effective neuronal function and survival. Lysosomes, which are membrane-bound organelles responsible for the degradation of macromolecules, are among the many cargoes being transported. Compartmentalized systems that allow for the separation of the somatic compartment from the axonal network, are widely used in the field of neurobiology and in the study of axonal transport in particular. Among the various solutions available, microfluidics chambers that take advantage of fluidic separation between different compartments, have seen widespread adoption. Said chambers are made of polydimethylsiloxane (PDMS), a transparent, gas permeable compound, which is compatible with fluorescence microscopy, and have significantly positively impacted cellular neuroscience, drastically increasing our understanding of axonal peripheral signaling. Here we describe a two-layered microfluidics chamber, engineered to allow for the culture of adult sensory neurons. This device was designed to promote the proper placement of adult sensory neurons in the somatic chamber in proximity of the microgrooves. We detail the production of the master mold, how to fabricate and assemble the device and how to disaggregate and load the cells in it. In addition, we provide details on how to conduct and analyze an axonal transport experiment using a custom made script in MATLAB designed by our laboratory.

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Acknowledgments

We thank the OIST Imaging Section for providing access to Nikon A1R laser confocal system and a scanning electron microscope, and the Mechanical Engineering and Microfabrication Support Section for their support in the MFC generation. We thank Prof. Amy Shen for fruitful discussion about the MFC design. M.F.E. and M.T. participated in the design and writing of this method paper. M.F.E., M.O., P.B., and M.T. collected and analyzed the experimental data. L.A. designed and implemented the script for axonal transport quantification . This work was generously funded by JSPS/Kakenhi #20K07458 to M.T. The authors declare not to have any conflict of interests.

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Authors and Affiliations

  1. Molecular Neuroscience Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

    Maria Fransiska Emily, Lokesh Agrawal, Miki Otsuki & Marco Terenzio

  2. Imaging Section, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan

    Paolo Barzaghi

Authors
  1. Maria Fransiska Emily
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  2. Lokesh Agrawal
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  3. Paolo Barzaghi
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  4. Miki Otsuki
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  5. Marco Terenzio
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Corresponding author

Correspondence to Marco Terenzio .

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Editors and Affiliations

  1. Department of Basic and Clinical Neurosciences, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK

    Alessio Vagnoni

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Emily, M.F., Agrawal, L., Barzaghi, P., Otsuki, M., Terenzio, M. (2022). Use of Microfluidics Chambers to Image Axonal transport in Adult Sensory Neurons. In: Vagnoni, A. (eds) Axonal Transport. Methods in Molecular Biology, vol 2431. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1990-2_14

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  • DOI: https://doi.org/10.1007/978-1-0716-1990-2_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1989-6

  • Online ISBN: 978-1-0716-1990-2

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