Name: studying neurobehavioral effects of environmental pollutants on zebrafish larvae
Uploaded: 2020-02-05T15:00:00
Description: Watch this Scientific Journal Video about Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae at JoVE.com

The authors are grateful for the financial support by the National Natural Science Foundation of China (21876135 and 21876136), the National Major Science and Technology Project of China (2017ZX07502003-03, 2018ZX07701001-22), the Foundation of MOE-Shanghai Key Laboratory of Children&#39;s Environmental Health (CEH201807-5), and Swedish Research Council (No. 639-2013-6913).

The protocol is in accordance with guidelines approved by the Animal Ethics Committee of Tongji University.
1. Zebrafish embryo collection
<ol>
	<li>Put two pairs of healthy adult Tubingen zebrafish into the spawning box on the night before exposure, keeping the sex ratio at 1:1.</li>
	<li>Remove the adult fish back to the system 30-60 min after daylight the next morning.</li>
	<li>Remove the embryos out of the spawning box.</li>
	<li>Rinse the embryos with system water.</li>
	<li>Transfer t...

<ol>
	<li>Sun, L., 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=Developmental+neurotoxicity+of+organophosphate+flame+retardants+in+early+life+stages+of+Japanese+medaka+(Oryzias+latipes).">Developmental neurotoxicity of organophosphate flame retardants in early life stages of Japanese medaka (Oryzias latipes).</a> Environmental Toxicology and Chemistry. 35 (12), 2931-2940 (2016).</li><li>Tian, L., 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=Neurotoxicity+induced+by+zinc+oxide+nanoparticles:+age-related+differences+and+interaction.">Neurotoxicity induced by zinc oxide nanoparticles: age-related differences and interaction.</a> Scientific Reports. 5, 16117 (2015).</li><li>Rauh, V. A., Margolis, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Environmental Organic Chemistry. , (2016).</li><li>Akortia, E., 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=A+review+of+sources,+levels,+and+toxicity+of+polybrominated+diphenyl+ethers+(PBDEs)+and+their+transformation+and+transport+in+various+environmental+compartments.">A review of sources, levels, and toxicity of polybrominated diphenyl ethers (PBDEs) and their transformation and transport in various environmental compartments.</a> Environmental Reviews. 24 (3), 253-273 (2016).</li><li>Shaw, B. J., Liddle, C. C., Windeatt, K. M., Handy, R. 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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locomotor+activity+changes+on+zebrafish+larvae+with+different+2,+2?,+4,+4?-tetrabromodiphenyl+ether+(PBDE-47)+embryonic+exposure+modes.">Locomotor activity changes on zebrafish larvae with different 2, 2?, 4, 4?-tetrabromodiphenyl ether (PBDE-47) embryonic exposure modes.</a> Chemosphere. 94, 53-61 (2014).</li><li>Zhang, B., 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=Neurobehavioral+effects+of+two+metabolites+of+BDE-47+(6-OH-BDE-47+and+6-MeO-BDE-47)+on+zebrafish+larvae.">Neurobehavioral effects of two metabolites of BDE-47 (6-OH-BDE-47 and 6-MeO-BDE-47) on zebrafish larvae.</a> Chemosphere. 200, 30-35 (2018).</li><li>Yang, X., 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=The+chlorine+contents+and+chain+lengths+influence+the+neurobehavioral+effects+of+commercial+chlorinated+paraffins+on+zebrafish+larvae.">The chlorine contents and chain lengths influence the neurobehavioral effects of commercial chlorinated paraffins on zebrafish larvae.</a> Journal of Hazardous Materials. 377, 172-178 (2019).</li><li>Schmitt, C., McManus, M., Kumar, N., Awoyemi, O., Crago, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analyses+of+the+neurobehavioral,+molecular,+and+enzymatic+effects+of+organophosphates+on+embryo-larval+zebrafish+(Danio+rerio).">Comparative analyses of the neurobehavioral, molecular, and enzymatic effects of organophosphates on embryo-larval zebrafish (Danio rerio).</a> Neurotoxicology and Teratology. 73, 67-75 (2019).</li><li>Li, X., Kong, H., Ji, X., Gao, Y., Jin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+behavioral+phenomics+applied+for+phenotyping+aquatic+neurotoxicity+induced+by+lead+contaminants+of+environmentally+relevant+level.">Zebrafish behavioral phenomics applied for phenotyping aquatic neurotoxicity induced by lead contaminants of environmentally relevant level.</a> Chemosphere. 224, 445-454 (2019).</li><li>Leuthold, D., Klu&#776;ver, N., Altenburger, R., Busch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+environmentally+relevant+neuroactive+chemicals+specifically+be+detected+with+the+locomotor+response+test+in+zebrafish+embryos?.">Can environmentally relevant neuroactive chemicals specifically be detected with the locomotor response test in zebrafish embryos?.</a> Environmental Science &amp; Technology. 53 (1), 482-493 (2018).</li><li>Kinch, C. D., Ibhazehiebo, K., Jeong, J. H., Habibi, H. R., Kurrasch, D. 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Effect of swimming speed.</a> Journal of Experimental Biology. 207 (19), 3265-3279 (2004).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> The Zebrafish Book. 4, (2000).</li><li>Ying, L., Jiang, L., Bo, P., Yong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogenic+effects+of+embryonic+exposure+to+pretilachlor+on+the+larvae+of+zebrafish.">Teratogenic effects of embryonic exposure to pretilachlor on the larvae of zebrafish.</a> Journal of Agro-Environment Science. 36 (3), 481-486 (2017).</li><li>Macphail, R. C., 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=Locomotion+in+larval+zebrafish:+Influence+of+time+of+day,+lighting+and+ethanol.">Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol.</a> Neurotoxicology. 30 (1), 52-58 (2009).</li><li>Kais, B., 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=DMSO+modifies+the+permeability+of+the+zebrafish+(Danio+rerio)+chorion-implications+for+the+fish+embryo+test+(FET).">DMSO modifies the permeability of the zebrafish (Danio rerio) chorion-implications for the fish embryo test (FET).</a> Aquatic Toxicology. 140, 229-238 (2013).</li><li>Truong, L., Harper, S. L., Tanguay, R. 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This work provides a detailed experimental protocol to evaluate the neurotoxicity of environmental pollutants using zebrafish larvae. Zebrafish go through the process from embryos to larvae during the exposure period, which means that good care of the embryos and larvae is essential. Anything that affects the development of the embryos and larvae can influence the final result. Here the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the whole assay.
<p class=...

In recent years more and more environmental pollutants have been proved neurotoxic1,2,3,4. However, the assessment of neurotoxicity in vivo after exposure to environmental pollutants is not as easy as that of endocrine disruption or developmental toxicity. In addition, early exposure to pollutants, especially at environmentally relevant doses, has attracted increasing attention in toxicity studies5,6,7,8.
Zebrafish is being established as an animal model fit for neurotoxicity studies during early development after exposure to environmental pollutants. Zebrafish are vertebrates that develop faster than other species after fertilization. The larvae do not need to be fed because the nutrients in the chorion are enough for sustain them for 7 days postfertilization (dpf)9. Larvae come out from the chorion at ~2 dpf and develop behaviors such as swimming and turning that can be observed, tracked, quantified, and analyzed automatically using behavior instruments10,11,12,13 starting at 3-4 dpf14,15,16,17,18. In addition, high-throughput tests can also be realized by behavior instruments. Thus, zebrafish larvae are an outstanding model for the neurobehavioral study of environmental pollutants19. Here, a protocol is offered using high-throughput monitoring to study the neurobehavioral toxicity of environmental pollutants on zebrafish larvae under light stimuli.
Our lab has studied the neurobehavioral toxicity of 2,2&#39;,4,4&#39;-tetrabromodiphenyl ether (BDE-47)20,21, 6&#39;-Hydroxy/Methoxy-2,2&#39;,4,4&#39;-tetrabromodiphenyl ether (6-OH/MeO-BDE-47)22, deca-brominated diphenyl ether (BDE-209), lead, and commercial chlorinated paraffins23 using the presented protocol. Many labs also use the protocol to study the neurobehavioral effects of other pollutants on larvae or adult fish24,25,26,27. This neurobehavioral protocol was used to help provide mechanistic support showing that low-dose exposure to bisphenol A and replacement bisphenol S induced premature hypothalamic neurogenesis in embryonic zebrafish27. In addition, some researchers optimized the protocol to perform corresponding studies. A recent study eliminated the toxicity of amyloid beta (A&#946;) in an easy, high-throughput zebrafish model using casein-coated gold nanoparticles (&#946;Cas AuNPs). It showed that &#946;Cas AuNPs in systemic circulation translocated across the blood-brain barrier of zebrafish larvae and sequestered intracerebral A&#946;42, eliciting toxicity in a nonspecific, chaperone-like manner, which was supported by behavioral pathology28.
Locomotion, path angle, and social activity are three neurobehavioral indicators used to study the neurotoxicity effects of zebrafish larvae after exposure to pollutants in the presented protocol. Locomotion is measured by the swimming distance of larvae and can be damaged after exposure to pollutants. Path angle and social activity are more closely related with the function of the brain and the central nervous system29. The path angle refers to the angle of the path of animal motion relative to the swimming direction30. Eight angle classes from ~-180&#176;-~+180&#176; are set in the system. To simplify the comparison, six classes in the final outcome are defined as routine turns (-10&#176; ~0&#176;, 0&#176; ~+10&#176;), average turns (-10&#176; ~-90&#176;, +10&#176; ~+90&#176;), and responsive turns (-180&#176; ~-90&#176;, +90&#176; ~+180&#176;) according to our previous studies21,22. Two-fish social activity is fundamental of group shoaling behavior; here a distance of &#60; 0.5 cm between two larvae valid is defined as social contact.
The protocol presented here demonstrates a clear process for studying neurobehavioral effects on zebrafish larvae and provides a way to reveal the neurotoxicity effects of various substances or pollutants. The protocol will benefit researchers interested in studying the neurotoxicity of environmental pollutants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48-well-microplate</td>
			<td>Corning</td>
			<td>3548</td>
			<td>Embyros housing</td>
		</tr>
		<tr>
			<td>6-well-microplate</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Embyros housing</td>
		</tr>
		<tr>
			<td>BDE-47</td>
			<td>AccuStandard</td>
			<td>5436-43-1</td>
			<td>Pollutant</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>67-68-5</td>
			<td>Cosolvent</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>SZX 16</td>
			<td>Observation instrument</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000267</td>
			<td>Transfer solution</td>
		</tr>
		<tr>
			<td>Zebrabox</td>
			<td>Viewpoint</td>
			<td>ZebraBox</td>
			<td>Behavior instrument</td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>Shanghai FishBio Co., Ltd.</td>
			<td>Tubingen</td>
			<td>Zebrafish supplier</td>
		</tr>
		<tr>
			<td>ZebraLab</td>
			<td>Viewpoint</td>
			<td>ZebraLab</td>
			<td>Behavior software</td>
		</tr>
	</tbody>
</table>

studying neurobehavioral effects of environmental pollutants on zebrafish larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

Here, we describe a protocol for studying the neurobehavioral effects of environmental pollutants using zebrafish larvae under light stimuli. The locomotion, path angle, and social activity tests are defined in the introduction. The setup of the microplates in the locomotion and path angle tests and the images of the software are shown below. In addition, our own research results are presented as examples. Two studies present the locomotion and path angle effects after exposure to BDE-47 ...

Watch this Scientific Journal Video about Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae at JoVE.com

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Our protocol provides a clear process for studying the neurobehavioral effects of the substances and the pollutants of interest on zebrafish larvae and for revealing the potential neurotoxicity of these agents. The technique can be performed using high throughput testing of neurobehaviors in the zebrafish model. This method can be applied to evaluate allele-neurobehavioral effects of pollutants to demonstrate potential neurotoxicity on us humans, as zebrafish genome has a high homology with humans, so it can provide indications for chemical management and the public health problems caused by pollutants.As zebrafish has the advantage of high throughput screening, this method provides insight into both development of psychotropic drugs and environmental toxicology research. Don't be intimidated about studying the first experiment. If you follow the protocol, you will have a successful outcome.At least two hours before performing an experiment, set the room temperature to 28 degrees Celsius and add 800 microliters of exposure solution to each well of a 48-well microplate. Then, use a one-milliliter pipette with a modified tip to transfer one larva in 200 microliters of exposure solution to each well of the microplate in preparation for a locomotion and path angle experiment. To prepare a six-well microplate for social behavior experiments, add four milliliters of exposure solution to each well of the six well microplate and transfer two larvae in 200 microliters of exposure solution to each well.Before beginning the test, open the high throughput monitoring software program and select the tracking rotations path angles module. Transfer the 48-well microplate to the recording platform and pull down the cover. Click File and Generate Protocol to begin generating a new protocol and enter 48 into the Location count position.Click Parameters, Protocol Parameters, and Time, set the Experiment duration to one hour and 10 minutes, and the Integration period to 60 seconds. Using the elliptical shape tool, draw a circle around the top left well. Select the circle and click Copy, Top Right Mark, Paste, and Select.Use the mouse to drag the copied circle to the top right well and click Copy, Top Right Mark, Paste, and Select again. Then, drag the copied circle to the bottom right well and click Build and Clear Marks. The system will automatically draw a circle over every other well of the plate.Next, click Draw Scale and draw a calibration line on the screen. Enter the length of the line, set the unit, and click Apply to Group. Set the animal color to black and set the detection threshold to 16 to 18 to allow detection of the animals, set the inactive to small speed to 0.5 centimeters per second and the small to large speed to 2.5 centimeters per second.Set the path angle classes from minus 180 to plus 180 as indicated. To set the light conditions, click Parameters, Light Driving, Uses one of the 3 triggering methods below, and Enhanced stimuli to set the light conditions. Click Edge and set a dark period of 10 minutes, followed by three cycles of alternating 10-minute light and dark periods.Then, save the protocol. When all of the parameters have been set, turn down the lighting in the test room, and allow the larvae to acclimate in the system for 10 minutes. At the end of the acclimation period, click Experiment and Execute, and create the folder in which the experimental files are to be saved.Then, enter the result name and click Background and Start. At the end of the experiment, click Experiment and Stop. Then return the 48-well microplate back to the light incubator for subsequent experiments.Because every group can include 16 animals, the system can be used to perform high throughput tests of the locomotion and path angle under different treatment conditions in a single 48-well microplate. The social activity of the larvae can be tested using six-well plates. In this representative experiment, the highest concentration group of BDE-47 demonstrated a pronounced hypoactivity during the dark period, while no changes due to BDE-47 exposure were observed during the light periods.Further, the high concentration group of 6-hydroxy-BDE-47 performed fewer routine and average turns at five days post-fertilization. However, more responsive turns were induced in the 6-methoxy-BDE47 exposure groups. The social activity of the zebrafish was stimulated by chlorinated paraffin-70 and the short-chain chlorinated paraffin-52b, while the long-chain chlorinated paraffin-52a shortened the duration for contact of the larvae.It's important to make sure that the transferred larvae for the test should have a normal swimming ability and no malformations. Other variables, such as color preference, light-dark preference, all darkness, tail movements of zebrafish, can be included to answer additional questions about neurobehavioral toxicity of environmental pollutants. The BDE-47 exposure solutions are neurotoxic and have endocrine disruption potential, so be sure to avoid direct contact with skin.

Research

JoVE Journal

Environment

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