ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H₂O₂) is best characterized with respect to roles in cellular signaling. H₂O₂ has been implicated during the development in several species. For example, a transient increase in H₂O₂ has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H₂O₂ source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in vivo and in vitro. Here, we describe a method for imaging intracellular H₂O₂ levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H₂O₂-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H₂O₂ levels at the tissue level. The sensor has been validated by the addition of H₂O₂. Additionally, this methodology could be used to measure H₂O₂ levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H₂O₂ during neuronal and general embryonic development in vertebrates.