Published: February 9th, 2021
This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general 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.
Reactive oxygen species (ROS) signaling regulates development and functioning of the nervous system1. An important cellular ROS source are NADPH oxidases (NOX), which are transmembrane proteins generating superoxide and hydrogen peroxide (H2O2)2. NOX enzymes are found throughout the central nervous system (CNS), and NOX-derived ROS contribute to neuronal development3,4,5,6. Maintenance and differentiation of neural stem cells, establishing neuronal polarity, neurite outgro....
All animal experiments were ethically reviewed and approved by the Purdue Animal Care and Use Committee (PACUC), following NIH guidelines with the protocol 2006002050 approved on 07/24/2020.
1. Preparation of solutions
Cultured zebrafish RGCs extend axons within 1d. A representative 405/480 ratio image of the H2O2-biosensor is shown in Figure 4A. The cell body, axon, and growth cones are clearly visible in individual neurons. These neurons can be subjected to different treatments over time to monitor H2O2 changes. We previously found that adding 100 µM H2O2 to culture media increases the ratio values, showing that real-time changes can .......
There are several critical steps that need attention throughout this protocol. We believe considering these points will improve the experimental flow. For primary RGC culture, the sterility of the ZFCM(-) is very important, since this culture media does not contain antibiotics and contamination can occur before or during imaging. To avoid it, we advise to prepare and use ZFCM(-) only inside a biosafety cabinet and make fresh ZFCM(-) media regularly (Step 1.5). In addition, laminin stocks should be kept at -80 °.......
This work was supported by the National Institutes of Health (Grant R01NS117701), National Science Foundation (Grant 1146944-IOS), the Indiana Traumatic Spinal Cord and Brain Injury Research Fund (Grant 20000289), the Purdue Research Foundation (Grant 209911), and the Office of the Executive Vice President for Research and Partnerships at Purdue University (Grant 210362). We thank Dr. Cory J. Weaver and Haley Roeder for establishing zebrafish RGC culture protocol. We thank Haley Roeder additionally for providing the data of Figure 4. We thank Leah Biasi and Kenny Nguyen for the help with RGC culture. We thank Gentry Lee for editing the text. We thank Dr. Tobias Dick f....
|35-mm culture dish
|35-mm glass bottom dish
|Borosilicate Glass Capillary Tubes
|Calcium Chloride Dihydrate
|Disposable Petri Dishes (100 x 15 mm)
|Fetal Bovine Serum
|Adaptive Science Tools
|Laser Scanning Confocal Microscopy
|Leibovitz's L-15 Medium with phenol red
|Leibovitz's L-15 Medium without phenol red
|Low melting agarose
|mMessage mMachine SP6 Transcription Kit
|New England Biolabs
|World Precision Instruments
|QiaQUICK PCR Purification Kit
|Recombinant mouse slit2
|Steritop 0.22 µm filter
|Vertical Pipette Puller
|David Kopf Instruments
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