ROS Live Cell Imaging During Neuronal Development

Summary

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.

Abstract

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.

Introduction

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 development^3,4,5,6. Maintenance and differentiation of neural stem cells, establishing neuronal polarity, neurite outgro....

Protocol

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

1. E2 media (1x)
   1. Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components shown in Table 1. Autoclave E2A, E2B and E2C solutions. Store at 4 °C.
   2. For 1x E2 media: Combine 5 mL of.......

Discussion

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 °.......

Materials

Name	Company	Catalog Number	Comments
35-mm culture dish	Sarstedt	83-3900
35-mm glass bottom dish	MatTek	P35G-1.5-10-C
Agarose	Fisher Scientific	BP160-500
Borosilicate Glass Capillary Tubes	Sutter/Fisher Scientific	NC9029378
Calcium Chloride Dihydrate	Fisher Scientific	C79-500
Cover glass	Corning	2850-22
Disposable Petri Dishes (100 x 15 mm)	VWR	25384-094
Fetal Bovine Serum	ThermoFisher Scientific	26140087
Glucose	Sigma Aldich	G7528
HEPES	Sigma Aldich	H4034
Injection Mold	Adaptive Science Tools	TU-1
Inverted Microscope	Nikon	TE2000
Laminin	ThermoFisher Scientific	23017-015
Laser Scanning Confocal Microscopy	Zeiss	710
Leibovitz's L-15 Medium with phenol red	Gibco/Fisher Scientific	11-415-064
Leibovitz's L-15 Medium without phenol red	Gibco/Fisher Scientific	21-083-027
Low melting agarose	Promega	V2111
mMessage mMachine SP6 Transcription Kit	Invitrogen	AM1340
NotI	New England Biolabs	R0189S
PBS	Hyclone/Fisher Scientific	SH3025601
Penicillin/streptomycin	ThermoFisher Scientific	15140122
Phenol Red	Sigma Aldich	P0290
Phenylthiourea (PTU)	Sigma Aldich	P7629
Pneumatic PicoPump	World Precision Instruments	PV820
Poly-D-Lysine (PDL)	Sigma Aldich	P7280
QiaQUICK PCR Purification Kit	QIAGEN	28104
Recombinant mouse slit2	R&D Systems	5444-SL-050
Sodium Pyruvate	Sigma Aldich	P5280
Steritop 0.22 µm filter	Millipore	S2GPT05RE
TE Buffer	Ambion	AM9860
Tricaine Methanesulfonate	Sigma Aldich	E10521
Vertical Pipette Puller	David Kopf Instruments	700C