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 the embryos into a glass Petri dish (9 cm diameter) with enough system water.</li>
	<li>Observe the embryos under the microscope and select healthy embryos for later exposure. 
	NOTE: Healthy embryos are usually transparent with light golden color under the microscope. The unhealthy embryos are usually pale and clumped together as observed under the microscope.</li>
</ol>
2. Preparation before exposure
<ol>
	<li>Prepare the Hanks&#39; solution according to the guidelines of the zebrafish book31. 
	NOTE: The Hanks&#39; solution includes 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3.</li>
	<li>Dilute the Hanks&#39; solution to 10% Hanks&#39; solution using sterile water.</li>
	<li>Add 1 mL of DMSO into 999 mL of 10% Hanks&#39; solution to make a control solution of 10% Hanks&#39; solution including 0.1% DMSO. 
	NOTE: The next steps use BDE-47 as an example of an exposure solution.</li>
	<li>Dissolve 5 mg of the neat BDE-47 in 1 mL of 100% DMSO to make a standard exposure solution of 5 mg/mL.</li>
	<li>Vortex the 5 mg/mL solution for 1 min to completely dissolve the BDE-47 in the DMSO.</li>
	<li>Transfer 10 &#181;L of the 5 mg/mL solution to a 12 mL brown glass bottle.</li>
	<li>Add sterilized water to a final volume of 10 mL to make the concentration of BDE-47 exposure solution 5 mg/L and DMSO ratio at 0.1%, then vortex for 1 min.</li>
	<li>Transfer 10 &#181;L and 100 &#181;L of the 5 mg/L solution into two 100 mL volumetric flasks respectively.</li>
	<li>Add 10% Hanks&#39; solution including 0.1% DMSO (prepared in step 2.3) to 100 mL to make the final concentrations of the BDE-47 exposure solutions 5 &#181;g/L and 50 &#181;g/L, respectively.</li>
	<li>Transfer the solutions into 100 mL brown glass bottles and store them at 4 &#176;C.</li>
</ol>
3. Exposure of embryos
<ol>
	<li>Transfer ~50 embryos into each of the three glass Petri dishes (6 cm diameter) 3-5 hours post fertilization (hpf).</li>
	<li>Use a 1 mL pipette tip to blot the system water around the embryos.</li>
	<li>Use a pipette to transfer the control and two BDE-47 exposure solutions (control, 5 &#181;g/L, 50 &#181;g/L) into the three glass Petri dishes, respectively.</li>
	<li>Shake the glass Petri dishes gently one by one to make the embryos disperse in the bottom of the plate.</li>
	<li>Put the glass Petri dishes into the light incubator under 28.5 &#176;C.</li>
	<li>Renew half of the exposure solutions every 24 h until 5 dpf.</li>
	<li>Check the dead embryos of every group on 1 dpf and 2 dpf and calculate the death rate.</li>
	<li>Check the incubated embryos of every group on 2 dpf and 3 dpf and calculate the hatchability.</li>
	<li>Check the deformity of the larvae every day after they come out from the chorion and calculate the deformity rate of every group. 
	NOTE: The deformity indicators include pericardial cyst, spinal curvature, tail curvature, among others factors32.</li>
</ol>
4. Preparation for the behavior test
<ol>
	<li>Prepare a 48 well microplate for the locomotion and path angle test and three 6 well microplates for the social activity test on the morning of 5 dpf.</li>
	<li>Transfer 800 &#181;L of exposure solution into every well of the 48 well microplate. 
	NOTE: Use 16 wells for every group (i.e., the control solution, 5 &#181;g/L, and 50 &#181;g/L group).</li>
	<li>Use a 1 mL pipette tip to transfer 200 &#181;L of exposure solution with one larva from the glass Petri dish into one well of the 48 well microplate.</li>
	<li>Transfer 4 mL of exposure solution into every well of the 6 well microplate. 
	NOTE: Use one 6 well microplate for every group.</li>
	<li>Use a 1 mL pipette tip to transfer 200 &#181;L of exposure solution with two larvae into each well of the 6 well microplate. 
	NOTE: Every group has six repeating groups.</li>
	<li>Make sure the temperature of the test room is 28 &#176;C 2 h before the test. 
	NOTE: Behavioral tests are usually performed in the afternoon.</li>
</ol>
5. Behavioral test
<ol>
	<li>Locomotion and path angle test
	<ol>
		<li>Click the launcher icon on the computer desk to open the software (see Table of Materials) that controls the high-throughput monitoring enclosure to start the program.</li>
		<li>Choose the &#34;Tracking, Rotations, Path Angles&#34; module to enter the operating interface.</li>
		<li>Transfer the 48 well microplate prepared in step 4.3 to the recording platform and pull down the cover.</li>
		<li>Click the &#34;File&#34; &#34;Generate Protocol&#34; button in turn in the software to begin generating a new protocol.</li>
		<li>Input &#34;48&#34; in the &#34;Location count&#34; position and click the &#34;OK&#34; button.</li>
		<li>Click the &#34;Parameters&#34; &#34;Protocol Parameters&#34; &#34;Time&#34; button in turn in the software. Set the experiment duration for 1 h and 10 min and set the integration period to 60 s33.</li>
		<li>Draw the detected areas.
		<ol>
			<li>Select the elliptical shape and draw the first circle around the first top left well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Top-Right Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the top right well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Bottom Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the bottom right well.</li>
			<li>Click the &#34;Build&#34; &#34;Clear marks&#34; button in turn. 
			NOTE: The system will automatically draw every other well of the plate. The newly created areas should perfectly fit each well between the actual fish and its reflection on the side of the well.</li>
		</ol>
		</li>
		<li>Click the &#34;Draw Scale&#34; button, draw a calibration line on the screen (a diagonal path or parallel to the side of the microplate), enter its length and set the &#34;Unit&#34;. Then click the &#34;Apply to group&#34; button.</li>
		<li>Set the animal color at &#34;Black&#34; in the software.</li>
		<li>Set the detection threshold at 16-18 to allow the detection of the animals.</li>
		<li>Input inactive/small and small/large speed at 0.5 cm/s and 2.5 cm/s respectively.</li>
		<li>Set the path angle classes. Input &#34;-90, -30, -10, 0, 10, 30, and 90&#34; to make path angle classes from -180&#176;-+180&#176;.</li>
		<li>Set the light conditions
		<ol>
			<li>Click the &#34;Parameters&#34; &#34;Light driving&#34; &#34;Uses one of the 3 triggering methods below&#34; &#34;Enhanced stimuli&#34; button in turn to set the light conditions.</li>
			<li>Choose the &#34;Edge&#34; button, then set a dark period of 10 min, followed by three cycles of alternating 10 min light and dark periods.</li>
		</ol>
		</li>
		<li>Save the protocol and turn down the light of the test room.</li>
		<li>Acclimate the larvae in the system for 10 min and click the &#34;Experiment&#34; &#34;Execute&#34; button in turn, then choose the folder where the experiment files are saved and enter the result name.</li>
		<li>Click the &#34;Background&#34; &#34;Start&#34; button in turn to start the test.</li>
		<li>Click the &#34;Experiment&#34; &#34;Stop&#34; button in turn to stop the experiment when the test ends. 
		NOTE: The system shows the data tested when the system stops. The data include the tracked distance at three speed classes and path angle numbers at eight angle classes of every minute. For the locomotion test in the presented example, the total distance in every light period (10 min) is calculated and the difference between the control group and the treatment groups compared.</li>
		<li>Transfer the 48 well microplate back to the light incubator for other experiments.</li>
	</ol>
	</li>
	<li>Social activity test
	<ol>
		<li>Click on the launcher icon on the computer desk to open the software that controls the high-throughput monitoring enclosure to start the program.</li>
		<li>Choose the &#34;Social Interactions&#34; module to enter the operating interface.</li>
		<li>Transfer the prepared 6 well microplate (control group) in step 4.4 to the recording platform and pull down the cover.</li>
		<li>Click the &#34;File&#34; &#34;Generate Protocol&#34; button in turn in the software to begin generating a new protocol.</li>
		<li>Input &#34;6&#34; in the &#34;Location count&#34; position and click the &#34;OK&#34; button.</li>
		<li>Click the &#34;Parameters&#34; &#34;Protocol Parameters&#34; &#34;Time&#34; button in turn in the software. Set the experiment duration for 1 h and 10 min and set the integration period to 60 s.</li>
		<li>Draw the detected areas.
		<ol>
			<li>Select the elliptical shape and draw the first circle around the first top left well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Top-Right Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the top-right well.</li>
			<li>Select the circle, click the &#34;Copy&#34; &#34;Bottom Mark&#34; &#34;Paste&#34; &#34;Select&#34; button in turn, and use the mouse to drag the copied circle to the bottom right well.</li>
			<li>Click the &#34;Build&#34; &#34;Clear marks&#34; button in turn. 
			NOTE: The newly created areas should fit each well perfectly and between the actual larvae and its reflection on the side of the well.</li>
		</ol>
		</li>
		<li>Click the &#34;Draw Scale&#34; button, draw a calibration line in the screen (a diagonal path or parallel to the side of the microplate), enter its length and set the &#34;Unit&#34;. Then click the &#34;Apply to group&#34; button.</li>
		<li>Set the detection threshold at 16-18 to allow the detection of the animals.</li>
		<li>Click the &#34;Black animal&#34; button in the software.</li>
		<li>Choose the &#34;Distance Threshold&#34; button and input &#34;5&#34; in the software.</li>
		<li>Set the light conditions.
		<ol>
			<li>Click the &#34;Parameters&#34; &#34;Light driving&#34; &#34;Uses one of the 3 triggering methods below&#34; &#34;Enhanced stimuli&#34; button in turn to set the light conditions.</li>
			<li>Choose the &#34;Edge&#34; button, then set a dark period of 10 min, followed by three cycles of alternating 10 min light and dark periods.</li>
		</ol>
		</li>
		<li>Save the protocol and turn down the light of the room.</li>
		<li>Acclimate the larvae in the system for 10 min and click the &#34;Experiment&#34; &#34;Execute&#34; button in turn, then choose the folder where the experiment files are saved and enter the result name.</li>
		<li>Click the &#34;Background&#34; &#34;Start&#34; button in turn to start the test.</li>
		<li>Click the &#34;Experiment&#34; &#34;Stop&#34; button in turn to stop the experiment in the software when the test ends. 
		NOTE: The system shows the data tested when the system stops.</li>
		<li>Transfer the 6 well microplate (control group) back to the light incubator for other experiments.</li>
		<li>Transfer the 6 well microplates (5 &#181;g/L and 50 &#181;g/L groups) in turn to the recording platform and repeat the steps from 5.2.4 to 5.2.17 by ordinal.</li>
	</ol>
	</li>
</ol>
6. Data analysis
<ol>
	<li>Open the spreadsheet file in the locomotion and path angle results.</li>
	<li>Select the three distance columns (inadist, smldist, lardist) and add them up. 
	NOTE: The data of inadist, smldist, and lardist mean different distances recorded by the system in different speed classes (inactive/small/large), respectively.</li>
	<li>For every 10 minute light period sum up the distance of every well, calculate the average distance of 16 wells, and compare the data of the three groups under light stimuli.</li>
	<li>For every 10 minute light period sum up the angle number of every well in every light duration from cl01 to cl08 in turn, and compare the data of the three groups under light stimuli. 
	NOTE: The data of columns from cl01 to cl08 mean different path angle numbers recorded by the system in different path angles, respectively.</li>
	<li>Open the spreadsheet file in the social activity results.</li>
	<li>Select the contct and contdur columns, and for every 10 min light period sum up the social times and their duration for every well.</li>
	<li>Calculate the average social times and duration of one group in every light duration and compare the data of the three groups under light stimuli.</li>
</ol>

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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=Low-dose+exposure+to+bisphenol+A+and+replacement+bisphenol+S+induces+precocious+hypothalamic+neurogenesis+in+embryonic+zebrafish.">Low-dose exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic neurogenesis in embryonic zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (5), 1475-1480 (2015).</li><li>Javed, I., 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=Inhibition+of+amyloid+beta+toxicity+in+zebrafish+with+a+chaperone-gold+nanoparticle+dual+strategy.">Inhibition of amyloid beta toxicity in zebrafish with a chaperone-gold nanoparticle dual strategy.</a> Nature Communications. 10 (1), 1-14 (2019).</li><li>Green, J., 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=Automated+high-throughput+neurophenotyping+of+zebrafish+social+behavior.">Automated high-throughput neurophenotyping of zebrafish social behavior.</a> Journal of Neuroscience Methods. 210 (2), 266-271 (2012).</li><li>Tytell, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamics+of+eel+swimming+II.+Effect+of+swimming+speed.">The hydrodynamics of eel swimming II. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drug Safety Evaluation. , 271-279 (2011).</li><li>Peeters, B. W., Moeskops, M., Veenvliet, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+preference+in+Danio+rerio:+effects+of+age+and+anxiolytic+treatments.">Color preference in Danio rerio: effects of age and anxiolytic treatments.</a> Zebrafish. 13 (4), 330-334 (2016).</li><li>Barba-Escobedo, P. A., Gould, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+social+preferences+of+lone+zebrafish+in+a+novel+environment:+strain+and+anxiolytic+effects.">Visual social preferences of lone zebrafish in a novel environment: strain and anxiolytic effects.</a> Genes, Brain and Behavior. 11 (3), 366-373 (2012).</li><li>Blaser, R., Penalosa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+affecting+zebrafish+(Danio+rerio)+behavior+in+the+light/dark+preference+test.">Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test.</a> Physiology &amp; Behavior. 104 (5), 831-837 (2011).</li><li>Blaser, R. E., Rosemberg, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+anxiety+in+zebrafish+(Danio+rerio):+dissociation+of+black/white+preference+and+novel+tank+test.">Measures of anxiety in zebrafish (Danio rerio): dissociation of black/white preference and novel tank test.</a> PloS One. 7 (5), e36931 (2012).</li><li>Weichert, F. G., Floeter, C., Artmann, A. S. M., Kammann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+ecotoxicity+of+potentially+neurotoxic+substances-Evaluation+of+a+behavioural+parameter+in+the+embryogenesis+of+Danio+rerio.">Assessing the ecotoxicity of potentially neurotoxic substances-Evaluation of a behavioural parameter in the embryogenesis of Danio rerio.</a> Chemosphere. 186, 43-50 (2017).</li></ol>

The authors have nothing to disclose.

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><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 ...

studying-neurobehavioral-effects-environmental-pollutants-on

Research

JoVE Journal

Environment

5.9K Views.  Tongji University. Ons protocol biedt een duidelijk proces voor het bestuderen van de neurogedragseffecten van de stoffen en de verontreinigende stoffen die van belang zijn voor zebravissenlarven en voor het onthullen van de potentiële neurotoxiciteit van deze stoffen. De techniek kan worden uitgevoerd met behulp van hoge doorvoer testen van neurobehaviors in de zebravis model. Deze methode kan worden toegepast om alleel-neurobehavioral effecten van verontreinigende stoffen te evalueren om potentiële neurotox...

Video: Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Behavioral Approaches to Studying Innate Stress in Zebrafish

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of ...

Behavior

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

Neuroscience

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

A Novel Method of Drug Administration to Multiple Zebrafish Danio rerio and the Quantification of Withdrawal

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Pharmacological Validation of the Prepulse Inhibition of Startle Response in Larval Zebrafish using a Commercial Automated System and Software

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, to the best of our knowledge, all PPI assays performed in the zebrafish have, until now, been done using custom made systems which were only available to individual groups. This has thereby presented challenges, particularly with regard to issues of data reproducibility and standardization. In the present work, we generated a protocol that utilizes commercially available automated systems to pharmacologically validate the PPI&#160;assay in larval zebrafish. Consistent with published findings, we were able to replicate the results of apomorphine, haloperidol and ketamine on the PPI response of 6 days post-fertilization zebrafish larvae.

Here we describe a protocol that utilizes commercially available automated systems to pharmacologically validate the prepulse inhibition (PPI) assay in larval zebrafish.

While there is an abundance of commercial and standardized automated systems and software for performing the prepulse inhibition (PPI) assay in rodents, ...

Brain Ventricular Microinjections of Lipopolysaccharide into Larval Zebrafish to Assess Neuroinflammation and Neurotoxicity

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research and develop alternative in vivo neuroinflammation models to understand the role of neuroinflammation in neurodegeneration. In this study, a larval zebrafish model of neuroinflammation mediated by ventricular microinjection of lipopolysaccharide (LPS) to induce an immune response and neurotoxicity was developed and validated. The transgenic zebrafish lines elavl3:mCherry, ETvmat2:GFP, and mpo:EGFP were used for real-time quantification of brain neuron viability by fluorescence live imaging integrated with fluorescence intensity analysis. The locomotor behavior of zebrafish larvae was recorded automatically using a video-tracking recorder. The content of nitric oxide (NO), and the mRNA expression levels of inflammatory cytokines including interleukin-6 (IL-6), interleukin-1&#946; (IL-1&#946;), and human tumor necrosis factor &#945; (TNF-&#945;) were investigated to assess the LPS-induced immune response in the larval zebrafish head. At 24 h after the brain ventricular injection of LPS, loss of neurons and locomotion deficiency were observed in zebrafish larvae. In addition, LPS-induced neuroinflammation increased NO release and the mRNA expression of IL-6, IL-1&#946;, and TNF-&#945; in the head of 6 days post fertilization (dpf) zebrafish larvae, and resulted in the recruitment of neutrophils in the zebrafish brain. In this study, injection of zebrafish with LPS at a concentration of 2.5-5 mg/mL at 5 dpf was determined as the optimum condition for this pharmacological neuroinflammation assay. This protocol presents a new, quick, and efficient methodology for brain ventricle microinjection of LPS to induce LPS-mediated neuroinflammation and neurotoxicity in a zebrafish larva, which is useful for studying neuroinflammation and could also be used as a high-throughput in vivo drug screening assay.

This protocol demonstrates the microinjection of lipopolysaccharide into the brain ventricular region in a zebrafish larval model to study the resulting neuroinflammatory response and neurotoxicity.

Neuroinflammation is a key player in various neurological disorders, including neurodegenerative diseases. Therefore, it is of great interest to research ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Biology

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

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