Axonal transport of organelles in motor neuron cultures using microfluidic chambers system

Topaz Altman, Roy Maimon, Ariel Ionescu, Tal Gradus Pery, Eran Perlson

Research output: Contribution to journalArticlepeer-review

Abstract

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Original languageEnglish
Article numbere60993
JournalJournal of Visualized Experiments
Volume2020
Issue number159
DOIs
StatePublished - May 2020

Keywords

  • Acidic compartments
  • Axonal transport
  • Issue 159
  • Microfluidic chambers
  • Mitochondria
  • Motor neurons
  • Neuroscience
  • Spinal cord explants

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