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Related Experiment Video

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ROS Live Cell Imaging During Neuronal Development
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ROS Live Cell Imaging During Neuronal Development.

Aslihan Terzi1, S M Sabbir Alam1, Daniel M Suter2

  • 1Department of Biological Sciences, Purdue University; Purdue Institute for Integrative Neuroscience, Purdue University.

Journal of Visualized Experiments : Jove
|March 1, 2021
PubMed
Summary

This study introduces a new method to visualize hydrogen peroxide (H2O2) in zebrafish neurons and embryos. This technique allows researchers to track H2O2 signaling during development and in specific cells.

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Area of Science:

  • Neuroscience
  • Developmental Biology
  • Biochemistry

Background:

  • Reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), are crucial signaling molecules in physiological processes.
  • H2O2 plays a role in embryonic development, with transient increases observed in zebrafish embryos.
  • NADPH oxidase (NOX)-derived H2O2 is essential for nervous system development, including retinal ganglion cell (RGC) differentiation and axonal growth.

Purpose of the Study:

  • To present a novel method for imaging intracellular H2O2 levels in zebrafish neurons and whole larvae.
  • To utilize a genetically encoded H2O2-specific biosensor, roGFP2-Orp1, for real-time monitoring.
  • To validate the biosensor's ratiometric readout for accurate H2O2 measurements, minimizing artifacts.

Main Methods:

  • Culture of zebrafish RGCs derived from embryos expressing the roGFP2-Orp1 biosensor.
  • Application of the roGFP2-Orp1 biosensor in whole zebrafish larvae to monitor tissue-level H2O2 dynamics.
  • Validation of the biosensor's response through controlled H2O2 addition.

Main Results:

  • Demonstration of successful imaging of intracellular H2O2 in cultured zebrafish RGCs.
  • Successful monitoring of H2O2 levels at the tissue level in whole zebrafish larvae during development.
  • Validation of the roGFP2-Orp1 biosensor's sensitivity and reliability in detecting H2O2.

Conclusions:

  • The roGFP2-Orp1 biosensor provides a robust method for visualizing H2O2 signaling in zebrafish.
  • This imaging approach can be applied to specific cell types and tissues via transgenic expression.
  • The methodology serves as a valuable tool for investigating the role of H2O2 in vertebrate neuronal and embryonic development.