Name: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish (Video) | JoVE
Uploaded: 2023-11-03T12:40:00
Description: Watch this Scientific Journal Video about Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish at JoVE.com

This work was supported by &#34;Agencia Estatal de Investigaci&#243;n&#34; from the Spanish Ministry of Science and Innovation (project PID2020-113371RB-C21), IDAEA-CSIC, Severo Ochoa Centre of Excellence (CEX2018-000794-S). Juliette Bedrossiantz was supported by a PhD grant (PRE2018-083513) co-financed by the Spanish Government and the European Social Fund (ESF).

Strict compliance with ethical standards guarantees the welfare and proper treatment of the zebrafish used for experimentation. All experimental procedures were carried out under the guidelines established by the Institutional Animal Care and Use Committees (CID-CSIC). The protocols and results presented below were performed under the license granted by the local government (agreement number 11336).
1. Animal housing for behavioral testing
<ol>
	<li>Perform all tests (presen...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

<ol>
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V., Stewart, A. 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+Protocols+for+Neurobehavioral+Research.">Zebrafish Protocols for Neurobehavioral Research.</a> Neuromethods. , (2012).</li><li>Kalueff, A. 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=Towards+a+comprehensive+catalog+of+zebrafish+behavior+1.0+and+beyond.">Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.</a> Zebrafish. 10 (1), 70-86 (2013).</li><li>Egan, R. 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=Understanding+behavioral+and+physiological+phenotypes+of+stress+and+anxiety+in+zebrafish.">Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.</a> Behavioural Brain Research. 205, 38-44 (2009).</li><li>. Social behavior in Zebrafish Available from: <a target="_blank" href="https://www.noldus.com/applications/social-behavior-zebrafish">https://www.noldus.com/applications/social-behavior-zebrafish</a> (2012)</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>Miller, N., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+shoaling+behaviour+in+zebrafish+(Danio+rerio).">Quantification of shoaling behaviour in zebrafish (Danio rerio).</a> Behavioural Brain Research. 184 (2), 157-166 (2007).</li><li>Landin, 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=Oxytocin+receptors+regulate+social+preference+in+zebrafish.">Oxytocin receptors regulate social preference in zebrafish.</a> Scientific Reports. 10 (1), 5435 (2020).</li><li>Ogi, 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=Social+preference+tests+in+zebrafish:+A+systematic+review.">Social preference tests in zebrafish: A systematic review.</a> Frontiers in Veterinary Science. 7, 590057 (2021).</li><li>Bedrossiantz, 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=A+zebrafish+model+of+neurotoxicity+by+binge-like+methamphetamine+exposure.">A zebrafish model of neurotoxicity by binge-like methamphetamine exposure.</a> Frontiers in Pharmacology. 12, 770319 (2021).</li><li>Hamilton, T. J., Krook, J., Szaszkiewicz, J., Burggren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoaling,+boldness,+anxiety-like+behavior+and+locomotion+in+zebrafish+(Danio+rerio)+are+altered+by+acute+benzo[a]pyrene+exposure.">Shoaling, boldness, anxiety-like behavior and locomotion in zebrafish (Danio rerio) are altered by acute benzo[a]pyrene exposure.</a> Science of the Total Environment. 774, 145702 (2021).</li><li>Kane, A. S., Salierno, J. D., Brewer, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+32.">Chapter 32.</a> Fish models in behavioral toxicology: Automated Techniques, Updates, and Perspectives Methods in Aquatic Toxicology. Volume2, (2005).</li><li>Faria, 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=Glyphosate+targets+fish+monoaminergic+systems+leading+to+oxidative+stress+and+anxiety.">Glyphosate targets fish monoaminergic systems leading to oxidative stress and anxiety.</a> Environment International. 146, 106253 (2021).</li><li>Maximino, C., Costa, B., Lima, 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+review+of+monoaminergic+neuropsychopharmacology+in+zebrafish,+6+years+later:+Towards+paradoxes+and+their+solution.">A review of monoaminergic neuropsychopharmacology in zebrafish, 6 years later: Towards paradoxes and their solution.</a> Current Psychopharmacology. 5 (2), 96-138 (2016).</li><li>Maximino, 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=Role+of+serotonin+in+zebrafish+(Danio+rerio)+anxiety:+Relationship+with+serotonin+levels+and+effect+of+buspirone,+WAY+100635,+SB+224289,+fluoxetine+and+para-chlorophenylalanine+(pCPA)+in+two+behavioral+models.">Role of serotonin in zebrafish (Danio rerio) anxiety: Relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and para-chlorophenylalanine (pCPA) in two behavioral models.</a> Neuropharmacology. 71, 83-97 (2013).</li><li>Faria, 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=Therapeutic+potential+of+N-acetylcysteine+in+acrylamide+acute+neurotoxicity+in+adult+zebrafish.">Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish.</a> Scientific Reports. 9 (1), 16467 (2019).</li><li>Homer, B. D., Solomon, T. M., Moeller, R. W., Mascia, A., DeRaleau, L., Halkitis, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+abuse+and+impairment+of+social+functioning:+A+review+of+the+underlying+neurophysiological+causes+and+behavioral+implications.">Methamphetamine abuse and impairment of social functioning: A review of the underlying neurophysiological causes and behavioral implications.</a> Psychological Bulletin. 134 (2), 301-310 (2008).</li><li>Linker, 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=Assessing+the+maximum+predictive+validity+for+neuropharmacological+anxiety+screening+assays+using+zebrafish.">Assessing the maximum predictive validity for neuropharmacological anxiety screening assays using zebrafish.</a> Neuromethods. 51, 181-190 (2011).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+alternative+methods+to+a+new+toxicology.">From alternative methods to a new toxicology.</a> European Journal of Pharmaceutics and Biopharmaceutics. 77 (3), 338-349 (2011).</li><li>Cachat, J. M., Kalueff, A., Cachat, 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=Video-Aided+Analysis+of+Zebrafish+Locomotion+and+Anxiety-Related+Behavioral+Responses.">Video-Aided Analysis of Zebrafish Locomotion and Anxiety-Related Behavioral Responses.</a> Zebrafish Neurobehavioral Protocols. Neuromethods. 51, (2011).</li><li>Rosemberg, D. 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=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS ONE. 6 (5), e19397 (2011).</li><li>Blaser, R., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+phenotyping+in+Zebrafish:+Comparison+of+three+behavioral+quantification+methods.">Behavioral phenotyping in Zebrafish: Comparison of three behavioral quantification methods.</a> Behavioral Research Methods. 38 (3), 456-469 (2006).</li><li>Cachat, 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=Three-dimensional+neurophenotyping+of+adult+zebrafish+behavior.">Three-dimensional neurophenotyping of adult zebrafish behavior.</a> PLoS ONE. 6 (3), e17597 (2011).</li><li>Cachat, J. 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=Deconstructing+adult+zebrafish+behavior+with+swim+trace+visualizations.">Deconstructing adult zebrafish behavior with swim trace visualizations.</a> Neuromethods. 51, 191-201 (2011).</li></ol>

Characteristic anxiety behaviors observed in NTT have been positively correlated with serotonin levels analyzed in brains21. For example, after exposure to para-chlorophenylalanine (PCPA), an inhibitor of 5-HT biosynthesis, fish exhibited positive geotaxis as well as decreased brain 5-HT levels22, results very similar to those obtained with METH. Therefore, the decrease in brain serotonin levels and the display of positive geotaxis in METH-exposed zebrafish suggests that th...

The zebrafish (Danio rerio) is a popular model vertebrate species for ecotoxicology, drug discovery, and safety pharmacology studies. Its low cost, well-established molecular genetic tools, and conservation of key physiological processes involved in the morphogenesis and maintenance of the nervous system make zebrafish an ideal animal model for neuroscience research, including neurobehavioral toxicology1,2. The main endpoint for evaluating the neurotoxicity of a chemical was, until recently, the presence of neuropathological effects. Lately, however, behavioral endpoints have been incorporated into neurotoxicological screening protocols, and these functional outcomes are now commonly used to identify and determine the potential neurotoxicity of chemicals3,4. Moreover, behavioral endpoints are highly relevant from an ecological point of view, as even a very mild behavioral change in fish could endanger the survival of the animal in natural conditions5.
One of the most used behavioral assays in adult zebrafish research is the novel tank test (NTT), which measures anxiety-like behavior6,7. In this assay, fish are exposed to novelty (fish are placed in an unfamiliar tank), a mild aversive stimulus and their behavioral responses are observed. NTT is used to assess basal locomotor activity, geotaxis, freezing, and erratic movements of fish, principally. Erratic8 is characterized by abrupt changes of direction (zigzagging) and repeated episodes of accelerations (darting). It is an alarm reaction and is usually observed before or after freezing episodes. Freezing behavior corresponds to a complete cessation of the fish&#39;s movements (except for opercular and ocular movements) while on the bottom of the tank, as distinguished from immobility caused by sedation, which causes hypolocomotion, akinesia, and sinking8. Freezing is usually related to a high state of stress and anxiety and is also part of submissive behavior. Complex behaviors are excellent indicators of the state of anxiety of animals. NTT has been shown to be sensitive to pharmacological and genetic manipulation9, making it a valuable tool for studying the neural basis of anxiety and related disorders.
Zebrafish are a highly social species, so we can measure a wide range of social behaviors. The shoaling test (ST) and the social preference test (SPT) are the most used assays to assess social behavior10. The ST measures the tendency of fish to group together11 by quantifying their spatial behavior and movement patterns. ST is useful for studying group dynamics, leadership, social learning, and understanding the social behavior of many fish species12. The SPT in adult zebrafish was adapted from Crawley&#39;s preference for social novelty test for mice13&#160;and quickly became a popular behavioral assay for the study of social interaction in this model species14. These two tests have also been adapted for use in drug screening assays and have shown promise for identifying novel compounds that modulate social behavior15,16.
In general, behavioral assays in adult zebrafish are powerful tools that can provide valuable information on the behavior mechanisms or the neurophenotypes of active compounds and abused drugs17. This protocol details how to implement these behavioral tools7 with basic material resources and how to apply them in toxicity assays to characterize the effects of a wide range of neuroactive compounds. In addition, we will see that the same tests can be applied to assess the neurobehavioral effects of acute exposure to a neuroactive compound (methamphetamine) but also to characterize these effects after chronic exposure to environmental concentrations of a pesticide (glyphosate).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aquarium Cube shape</td>
			<td>Blau Aquaristic</td>
			<td>7782025</td>
			<td>Cubic Panoramic 10 &#160;(10 L, 20 cm x 20 cm x 25 cm, 5 mm)</td>
		</tr>
		<tr>
			<td>Ethovision software</td>
			<td>Noldus</td>
			<td>Ethovision XT</td>
			<td>Version 12.0 or newer</td>
		</tr>
		<tr>
			<td>GigE camera</td>
			<td>Imaging Development Systems</td>
			<td>UI-5240CP-NIR-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 9.02</td>
			<td>GraphPad software Inc</td>
			<td>GraphPad Prism 9.02</td>
			<td>&#160;For Windows</td>
		</tr>
		<tr>
			<td>IDS camera manager</td>
			<td>Imaging Development Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED backlight illumination</td>
			<td>Quirumed</td>
			<td>GP-G2</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Software</td>
			<td>IBM</td>
			<td>IBM SPSS v26</td>
			<td></td>
		</tr>
		<tr>
			<td>uEye Cockpit software&#160;</td>
			<td>Imaging Development Systems</td>
			<td>version 4.90</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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

In this section, we will look at some possible applications of these behavioral tools in fish neurotoxicology. The following results correspond to the characterization of the acute or binge effects of methamphetamine (METH), a recreational drug, and the sub-chronic effects of glyphosate, one of the main herbicides found in aquatic ecosystems.
Characterization of a methamphetamine binge neurotoxicity model in adult zebrafish 
When evaluating the effect of 40 mg/L METH on N...

Watch this Scientific Journal Video about Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish at JoVE.com

Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish (Video) | JoVE

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.