Name: ros live cell imaging during neuronal development
Uploaded: 2021-02-09T14:30:00
Description: Watch this Scientific Journal Video about ROS Live Cell Imaging During Neuronal Development at JoVE.com

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 for providing roGFP2-Orp1 and Dr. Qing Deng for pCS2+ vector containing roGFP2-Orp1. Figure 2&#160;is created with Biorender.com.

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
<ol>
	<li>E2 media (1x)
	<ol>
		<li>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 &#176;C.</li>
		<li>For 1x E2 media: Combine 5 mL of...

<ol>
	<li>B&#243;rquez, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+role+of+redox+signaling+in+neuronal+development.">Dissecting the role of redox signaling in neuronal development.</a> Journal of Neurochemistry. 137 (4), 506-517 (2016).</li><li>Bedard, K., Krause, K. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NOX+family+of+ROS-generating+NADPH+oxidases:+Physiology+and+pathophysiology.">The NOX family of ROS-generating NADPH oxidases: Physiology and pathophysiology.</a> Physiological Reviews. 87 (1), 245-313 (2007).</li><li>Weaver, C. J., Leung, Y. F., Suter, D. 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T., Gonz&#225;lez-Billault, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+NADPH+oxidase+to+the+establishment+of+hippocampal+neuronal+polarity+in+culture.">Contribution of NADPH oxidase to the establishment of hippocampal neuronal polarity in culture.</a> Journal of Cell Science. 128 (16), 2989-2995 (2015).</li><li>Munnamalai, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+interactions+between+Nox2-type+NADPH+oxidase+and+the+F-actin+cytoskeleton+in+neuronal+growth+cones.">Bidirectional interactions between Nox2-type NADPH oxidase and the F-actin cytoskeleton in neuronal growth cones.</a> Journal of Neurochemistry. 130 (4), 526-540 (2014).</li><li>Kishida, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+toolbox+for+genetic+manipulation+of+zebrafish.">A molecular toolbox for genetic manipulation of zebrafish.</a> Advances in Genomics and Genetics. 5, 151-163 (2015).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments. (25), e1115 (2009).</li><li>Avdesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regular+care+and+maintenance+of+a+Zebrafish+(Danio+rerio)+laboratory:+An+introduction.">Regular care and maintenance of a Zebrafish (Danio rerio) laboratory: An introduction.</a> Journal of Visualized Experiments. (69), e4196 (2012).</li><li>Chen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+neuron+culture+for+nerve+growth+and+axon+guidance+studies+in+Zebrafish+(Danio+rerio).">Primary neuron culture for nerve growth and axon guidance studies in Zebrafish (Danio rerio).</a> PLoS One. 8 (3), 57539 (2013).</li><li>Zhang, L., Leung, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+of+zebrafish+embryonic+eye+tissues.">Microdissection of zebrafish embryonic eye tissues.</a> Journal of Visualized Experiments. (40), e2028 (2010).</li><li>Suter, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+neuronal+growth+cone+motility+and+duidance+in+vitro.">Live cell imaging of neuronal growth cone motility and duidance in vitro.</a> Cell Migration: Methods in Molecular Biology. , 65-86 (2011).</li><li>Weaver, C. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+EGSH+and+H2O2+with+roGFP2-based+redox+probes.">Measuring EGSH and H2O2 with roGFP2-based redox probes.</a> Free Radical Biology and Medicine. 51, 1943-1951 (2011).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenylthiourea+specifically+reduces+zebrafish+eye+size.">Phenylthiourea specifically reduces zebrafish eye size.</a> PLoS One. 7 (6), 40132 (2012).</li><li>Ermakova, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+fluorescent+genetically+encoded+indicator+for+intracellular+hydrogen+peroxide.">Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide.</a> Nature Communications. 5, 5222 (2014).</li><li>Oparka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+ROS+levels+using+CM-H2DCFDA+and+HyPer.">Quantifying ROS levels using CM-H2DCFDA and HyPer.</a> Methods. 109, 3-11 (2016).</li><li>Dickinson, B. C., Peltier, J., Stone, D., Schaffer, D. V., Chang, C. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein-based+redox+probes.">Fluorescent protein-based redox probes.</a> Antioxidants &amp; Redox Signaling. 13 (5), 621-650 (2010).</li><li>Panieri, E., Millia, C., Santoro, M. 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There are several critical steps that need&#160;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 &#176;...

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 outgrowth, and synaptic plasticity have been shown to require adequate levels of ROS7,8,9,10,11. On the other hand, uncontrolled production of ROS by NOXes contribute to neurodegenerative disorders including Alzheimer&#39;s Disease, multiple sclerosis, and traumatic brain injury12,13,14. Hence, production of physiologically relevant ROS is critical to maintaining healthy conditions.
Development of genetically encoded biosensors facilitated the detection of cellular ROS greatly. One important advantage of genetically encoded biosensors is the increased temporal and spatial resolution of the ROS signal, as these sensors can be specifically targeted to distinct locations. Redox-sensitive GFP (roGFP) is one type of such ROS biosensors. The roGFP2-Orp1 variant specifically detects H2O2 through its Orp1 domain, which is a glutathione peroxiredoxin family protein from yeast15,16. The oxidation of the Orp1 protein is transferred to roGFP2 to alter its conformation (Figure 1A). The probe exhibits two excitation peaks near 405 nm and 480 nm, and a single emission peak at 515 nm. Upon oxidation, the fluorescence intensity around excitation peaks changes: while 405 nm excitation increases, 480 nm excitation decreases. Thus, roGFP2-Orp1 is a ratiometric biosensor, and H2O2-levels are detected by the ratio of fluorescence intensities excited at two different wavelengths (Figure 1B). Overall, roGFP2-Orp1 is a versatile tool for ROS imaging that can be utilized efficiently in vivo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62165/62165fig01.jpg" /> 
Figure 1: Schematic representation and excitation spectra of roGFP2-Orp1. (A) Oxidant transfer occurs between Orp1 and roGFP2 in response to H2O2, leading to conformational changes in roGFP2. (B) The excitation spectra of the roGFP2-Orp1 exhibits two excitation peaks at 405 nm and 480 nm and single emission peak at 515 nm. Upon oxidation by H2O2, the 405 nm excitation increases while 480 nm excitation decreases. This results in a ratiometric readout for H2O2 presence. The figure has been modified from Bilan and Belousov (2017)16 and Morgan et al. (2011)25. <a href="https://www.jove.com/files/ftp_upload/62165/62165fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The Danio rerio (zebrafish) model system has several advantages for applying genetically encoded biosensors. The optical transparency of the embryos and larvae enables non-invasive in vivo&#160;imaging. New imaging tools are being developed to achieve higher resolution and deeper penetration17. Furthermore, there are established tools for genetic manipulation (ectopic mRNA expression, Tol2 transgenesis, etc.) and genome editing (TALENs, CRISPR/Cas9, etc.), which promotes the generation of transgenic animals18. As the zebrafish embryos develop outside of the mother, this system further allows easier access and manipulation of the embryos. For instance, one-cell stage injections and drug treatments can easily be done.
Here, we used zebrafish to transiently express the H2O2-specific biosensor roGFP2-Orp1 by injecting in vitro-transcribed mRNA. These embryos can be used for both in vitro imaging of cultured neurons and in vivo imaging (Figure 2). We describe a protocol for dissecting and plating retinal ganglion cells (RGCs) from zebrafish embryos followed by assessing H2O2-levels in cultured neurons. Then, we present a method for in vivo imaging of roGFP2-Orp1-expressing embryos and larvae using confocal microscopy. This approach not only allows to determine physiological H2O2-levels but also potential changes occurring in different developmental stages or conditions. Overall, this system provides a reliable method for detecting H2O2 in live cells and animals to study the role of H2O2 in development, health, and disease.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62165/62165fig03.jpg" /> 
Figure 2. Outline of the experimental approach. Briefly, after embryo collection, roGFP2-Orp1 mRNA is injected into the yolk of one-cell stage zebrafish embryos. Developing embryos can be used for both (A) in vitro and (B) in vivo imaging. (A) GFP-positive embryos are used to dissect retinas for RGC collection at 34 hpf. Dissociated RGCs are plated on PDL/laminin-coated coverslips in ZFCM (+) media. Growth cone imaging can be conducted as RGCs extend their axons after 6-24 h of plating. Cells can be subjected to different treatments to measure the potential changes in H2O2-levels. Here, we measured H2O2-levels in the growth cones of RGCs (red). (B) GFP positive embryos are used for in vivo imaging. At the desired age, embryos can be anesthetized and mounted on 35 mm glass bottom dishes for confocal imaging. Here, embryos are mounted ventrally for retinal imaging. Schematic shows retinal development in zebrafish. RGCs form ganglion cell layer (GCL), which is the innermost layer in retina. RGC axons develop into optic nerve to cross midline, forming optic chiasm. Then, RGC axons grow dorsally to make synapses in the optic tectum in the midbrain. <a href="https://www.jove.com/files/ftp_upload/62165/62165fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35-mm culture dish</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillary Tubes</td>
			<td>Sutter/Fisher Scientific</td>
			<td>NC9029378</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes (100 x 15 mm)</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140087</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection Mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscopy</td>
			<td>Zeiss</td>
			<td>710</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium with phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>11-415-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium without phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>21-083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting agarose</td>
			<td>Promega</td>
			<td>V2111</td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage mMachine SP6 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R0189S</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone/Fisher Scientific</td>
			<td>SH3025601</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylthiourea (PTU)</td>
			<td>Sigma Aldich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>World Precision Instruments</td>
			<td>PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma Aldich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>QiaQUICK PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant mouse slit2</td>
			<td>R&#38;D Systems</td>
			<td>5444-SL-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma Aldich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop 0.22 &#181;m filter</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Ambion</td>
			<td>AM9860</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine Methanesulfonate</td>
			<td>Sigma Aldich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>700C</td>
			<td></td>
		</tr>
	</tbody>
</table>

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&#160;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.

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 &#181;M H2O2 to culture media increases the ratio values, showing that real-time changes can ...

Watch this Scientific Journal Video about ROS Live Cell Imaging During Neuronal Development at JoVE.com

ROS Live Cell Imaging During Neuronal Development

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 ...

Research

JoVE Journal

Neuroscience

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