Este trabalho foi apoiado pela "Agencia Estatal de Investigación" do Ministério da Ciência e Inovação da Espanha (projeto PID2020-113371RB-C21), IDAEA-CSIC, Centro de Excelência Severo Ochoa (CEX2018-000794-S). Juliette Bedrossiantz foi apoiada por uma bolsa de doutoramento (PRE2018-083513) co-financiada pelo Governo espanhol e pelo Fundo Social Europeu (FSE).

Bateria de Testes Comportamentais para Avaliação de Efeitos Neurotóxicos em Peixe-Zebra Adulto

<ol>
	<li>Rald&#250;a, D., Pi&#241;a, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+zebrafish+assays+for+analyzing+drug+toxicity.">In vivo zebrafish assays for analyzing drug toxicity.</a> Expert Opinion on Drug Metabolism &amp; Toxicology. 10 (5), 685-697 (2014).</li><li>Faria, M., Prats, E., Bellot, M., Gomez-Canela, C., Rald&#250;a, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+modulation+of+serotonin+levels+in+zebrafish+larvae:+Lessons+for+identifying+environmental+neurotoxicants+targeting+the+serotonergic+system.">Pharmacological modulation of serotonin levels in zebrafish larvae: Lessons for identifying environmental neurotoxicants targeting the serotonergic system.</a> Toxics. 9 (6), 118 (2021).</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=Zebrafish+models+for+human+acute+organophosphorus+poisoning.">Zebrafish models for human acute organophosphorus poisoning.</a> Scientific Reports. 5, 15591 (2015).</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>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=Screening+anti-predator+behaviour+in+fish+larvae+exposed+to+environmental+pollutants.">Screening anti-predator behaviour in fish larvae exposed to environmental pollutants.</a> Science of the Total Environment. 714, 136759 (2020).</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=Acrylamide+acute+neurotoxicity+in+adult+zebrafish.">Acrylamide acute neurotoxicity in adult zebrafish.</a> Scientific Reports. 8 (1), 7918 (2018).</li><li>Kalueff, A. 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>

Os autores declaram que a pesquisa foi conduzida na ausência de quaisquer relações comerciais ou financeiras que pudessem ser interpretadas como um potencial conflito de interesses.

Comportamentos característicos de ansiedade observados nas NTT têm sido positivamente correlacionados com os níveis de serotonina analisados em cérebros21. Por exemplo, após a exposição à paraclorofenilalanina (PCPA), um inibidor da biossíntese de 5-HT, os peixes exibiram geotáxis positivos, bem como diminuição dos níveis cerebrais de 5-HT22, resultados muito semelhantes aos obtidos com a metanfetamina. Portanto, a diminuição nos níveis de serotonina cerebra...

O peixe-zebra (Danio rerio) é uma espécie de vertebrado modelo popular para estudos de ecotoxicologia, descoberta de drogas e farmacologia de segurança. Seu baixo custo, ferramentas genéticas moleculares bem estabelecidas e conservação dos principais processos fisiológicos envolvidos na morfogênese e manutenção do sistema nervoso fazem do peixe-zebra um modelo animal ideal para pesquisas em neurociências, incluindo toxicologianeurocomportamental1,2. O principal desfecho para avaliar a neurotoxicidade de um produto químico era, até recentemente, a presença de efeitos neuropatológicos. Ultimamente, no entanto, desfechos comportamentais têm sido incorporados em protocolos de triagem neurotoxicológica, e esses desfechos funcionais são agora comumente usados para identificar e determinar a potencial neurotoxicidade de substâncias químicas 3,4. Além disso, os desfechos comportamentais são altamente relevantes do ponto de vista ecológico, pois mesmo uma mudança comportamental muito leve em peixes poderia colocar em risco a sobrevivência do animal em condições naturais5.
Um dos ensaios comportamentais mais utilizados na pesquisa do peixe-zebra adulto é o novel tank test (NTT), que mede o comportamento semelhante à ansiedade 6,7. Neste ensaio, os peixes são expostos à novidade (os peixes são colocados em um tanque desconhecido), a um estímulo aversivo leve e suas respostas comportamentais são observadas. O NTT é utilizado para avaliar principalmente a atividade locomotora basal, geotaxis, congelamento e movimentos erráticos de peixes. A errática8 é caracterizada por mudanças bruscas de direção (ziguezagueamento) e episódios repetidos de acelerações (ousadia). É uma reação de alarme e geralmente é observada antes ou depois de episódios de congelamento. O comportamento de congelamento corresponde à cessação completa dos movimentos dos peixes (exceto os movimentos operculares e oculares) no fundo do tanque, diferentemente da imobilidade causada pela sedação, que causa hipolocomoção, acinesia e afundamento8. O congelamento geralmente está relacionado a um alto estado de estresse e ansiedade e também faz parte do comportamento submisso. Comportamentos complexos são excelentes indicadores do estado de ansiedade dos animais. As NTT têm se mostrado sensíveis à manipulação farmacológica e genética9, tornando-se uma ferramenta valiosa para o estudo das bases neurais da ansiedade e transtornos relacionados.
O peixe-zebra é uma espécie altamente social, por isso podemos medir uma ampla gama de comportamentos sociais. O teste de cardume (TS) e o teste de preferência social (TCP) são os ensaios mais utilizados para avaliar o comportamento social10. O TS mede a tendência dos peixes aagruparem-se 11 quantificando seu comportamento espacial e padrões de movimento. O TS é útil para estudar dinâmicas de grupo, liderança, aprendizagem social e compreensão do comportamento social de muitas espécies de peixes12. O TCP em zebrafish adultos foi adaptado do teste de preferência de Crawley por novidade social para camundongos13 e rapidamente se tornou um ensaio comportamental popular para o estudo da interação social nesta espécie modelo14. Esses dois testes também foram adaptados para uso em ensaios de triagem de drogas e têm se mostrado promissores na identificação de novos compostos que modulam o comportamento social15,16.
Em geral, ensaios comportamentais em peixes-zebra adultos são ferramentas poderosas que podem fornecer informações valiosas sobre os mecanismos comportamentais ou os neurofenótipos de compostos ativos e drogas abusadas17. Este protocolo detalha como implementaressas ferramentas comportamentais7 com recursos materiais básicos e como aplicá-las em ensaios de toxicidade para caracterizar os efeitos de uma ampla gama de compostos neuroativos. Além disso, veremos que os mesmos testes podem ser aplicados para avaliar os efeitos neurocomportamentais da exposição aguda a um composto neuroativo (metanfetamina), mas também para caracterizar esses efeitos após exposição crônica a concentrações ambientais de um pesticida (glifosato).

<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

A presença de efeitos neuropatológicos provou ser, por muitos anos, o principal desfecho para avaliar a neurotoxicidade de uma substância química. No entanto, nos últimos 50 anos, os efeitos de produtos químicos sobre o comportamento de espécies-modelo têm sido ativamente investigados. Progressivamente, desfechos comportamentais foram incorporados em protocolos de triagem neurotoxicológica, e esses desfechos funcionais são agora rotineiramente usados para identificar e determinar a potencial neurotoxicidade de produtos químicos. Ensaios comportamentais em peixes-zebra adultos fornecem um meio padronizado e confiável para estudar uma ampla gama de comportamentos, incluindo ansiedade, interação social, aprendizagem, memória e dependência. Ensaios comportamentais em peixes-zebra adultos normalmente envolvem colocar os peixes em uma arena experimental e registrar e analisar seu comportamento usando software de rastreamento de vídeo. Os peixes podem ser expostos a vários estímulos, e seu comportamento pode ser quantificado usando uma variedade de métricas. O novo teste do tanque é um dos testes mais aceitos e amplamente utilizados para estudar o comportamento semelhante à ansiedade em peixes. Os testes de cardume e preferência social são úteis no estudo do comportamento social do peixe-zebra. Este ensaio é particularmente interessante uma vez que o comportamento de todo o cardume é estudado. Estes ensaios têm se mostrado altamente reprodutíveis e sensíveis a manipulações farmacológicas e genéticas, tornando-os ferramentas valiosas para o estudo dos circuitos neurais e mecanismos moleculares subjacentes ao comportamento. Adicionalmente, estes ensaios podem ser utilizados na triagem de fármacos para identificar compostos que possam ser potenciais moduladores do comportamento.
Mostraremos neste trabalho como aplicar ferramentas comportamentais em neurotoxicologia de peixes, analisando o efeito da metanfetamina, uma droga recreativa, e do glifosato, um poluente ambiental. Os resultados demonstram a significativa contribuição dos ensaios comportamentais em peixes-zebra adultos para a compreensão dos efeitos neurotoxicológicos de poluentes ambientais e drogas, além de fornecer informações sobre os mecanismos moleculares que podem alterar a função neuronal.

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).

Autor em destaque: Avaliação de mudanças comportamentais ambientais e induzidas por drogas em peixes-zebra adultos 

Avaliação da Neurotoxicidade em Danio rerio Adulto utilizando uma Bateria de Testes Comportamentais em um Único Tanque

Our main research aims to identify the behavioral effects induced by environmental pollutants and drugs in fish. Also trying to understand the mechanisms leading to these effects. A social and anxiety-like behavior cannot be conveniently addressed using embryos.We conduct studies targeting these behaviors in the adult Zebrafish model. This protocol details how to implement behavioral tools with basic material resources to facilitate the reproducibility of tests and harmonize the results obtained within the scientific community. The assays presented in this protocol have proven to be highly reproducible and sensitive to genetic and pharmacological manipulations, making them variable and noninvasive tools fostering the neural and molecular mechanisms underlying behavior.Once a behavioral effort has been identified, the most challenging part is to determine the specific modes of action leading to the observative efforts. Starting by the molecular initiating event where predictions can provide some clues on the molecular initiating event, we are still far from obtaining accurate predictions. We are currently interested in the determining the phase validity of the chemical models of Parkinson's disease developed in embryonic and adult Zebrafish by using MPTP and rotenone with the same, the kinematic of the gait-like movement or during an explosive movement like the acoustic evoked escape response, will be evaluated.

Nesta seção, veremos algumas possíveis aplicações dessas ferramentas comportamentais na neurotoxicologia de peixes. Os resultados a seguir correspondem à caracterização dos efeitos agudos ou compulsivos da metanfetamina (METH), uma droga recreativa, e dos efeitos subcrônicos do glifosato, um dos principais herbicidas encontrados em ecossistemas aquáticos.
Caracterização de um modelo de neurotoxicidade da compulsão por metanfetamina em zebrafish adulto Ao avalia...

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

Aqui, apresentamos uma bateria de teste comportamental abrangente, incluindo o novo tanque, Shoaling e testes de preferência social, para determinar efetivamente os potenciais efeitos neurotóxicos de produtos químicos (por exemplo, metanfetamina e glifosato) em peixes-zebra adultos usando um único tanque. Este método é relevante para neurotoxicidade e pesquisa ambiental.

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

Nossa pesquisa principal visa identificar os efeitos comportamentais induzidos por poluentes ambientais e drogas em peixes. Também tentando entender os mecanismos que levam a esses efeitos. Um comportamento social e ansioso não pode ser convenientemente abordado usando embriões.Realizamos estudos visando esses comportamentos no modelo adulto de Zebrafish. Este protocolo detalha como implementar ferramentas comportamentais com recursos materiais básicos para facilitar a reprodutibilidade dos testes e harmonizar os resultados obtidos na comunidade científica. Os ensaios apresentados neste protocolo mostraram-se altamente reprodutíveis e sensíveis a manipulações genéticas e farmacológicas, tornando-os ferramentas variáveis e não invasivas que favorecem os mecanismos neurais e moleculares subjacentes ao comportamento.Uma vez identificado um esforço comportamental, a parte mais desafiadora é determinar os modos específicos de ação que levam aos esforços observativos. Começando pelo evento de iniciação molecular, onde as previsões podem fornecer algumas pistas sobre o evento de iniciação molecular, ainda estamos longe de obter previsões precisas. Atualmente estamos interessados em determinar a validade de fase dos modelos químicos da doença de Parkinson desenvolvidos em peixes-zebra embrionários e adultos usando MPTP e rotenona com o mesmo, a cinemática do movimento de marcha ou durante um movimento explosivo como a resposta acústica de escape evocado, será avaliado.

neurotoxicity-assessment-adult-danio-rerio-using-battery-behavioral

Research

JoVE Journal

Neuroscience

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

1.1K visualizações.  Institute for Environmental Assessment and Water Research (IDAEA-CSIC). Nossa pesquisa principal visa identificar os efeitos comportamentais induzidos por poluentes ambientais e drogas em peixes. Também tentando entender os mecanismos que levam a esses efeitos. Um comportamento social e ansioso não pode ser convenientemente abordado usando embriões.Realizamos estudos visando esses comportamentos no modelo adulto de Zebrafish. Este protocolo detalha como implementar ferramentas comportamentais com recursos materiais básicos para facilitar a reprodutibil...

Vídeo: Autor em destaque: Avaliação de mudanças comportamentais ambientais e induzidas por drogas em peixes-zebra adultos 

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.

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.

Neurociência

Experimental Tank Configuration and Video Recording Setup for Behavioral Testing in Adult Zebrafish

To begin, assemble the square experimental tanks, cameras, and computers. For individual behavior assays of zebra fish. Fill the tank with seven liters of 28 degrees Celsius well-oxygenated fish water.Adjust the position of the tank in front of the camera to avoid distorted images. Using the LED backlight, check the illumination setup, and turn on the camera. Next, open the camera manager to check the availability of the jiggy camera on the computer.Launch the jiggy camera controlling software and open the Camera option. Then select Monochrome Mode and adjust the image size. Open camera properties, and under Camera, set the pixel clock to maximum and frame rate to 30 frames per second.Then, adjust the exposure. Now under Image, set the gain to zero and adjust the black levels. In the Size tab, adjust the window dimensions to the region requiring engraving.Once done, close the camera properties. Next, create a general folder for the experiment session to save the camera settings and videos. Then click File, Save Parameters, and to file to save the settings.After configuring all cameras, open record video sequence and select Create to save as a new video file. Select max frames, and enter 10, 800 in the frame box. Then select Calculate Frame Rate.Introduce the fish one by one at the bottom of the experimental tanks for behavioral tests, and click Record to start the recording. For the shoaling test, introduce the shoal at the bottom of the experimental tank. For the social preference test, introduce the fish one by one at the bottom of the different experimental tanks.Once a chat box with a maximum number of frames achieved appears on the screen, click Accept. Then select Close to conclude the recording. Finally, remove the fish recorded for behavioral tests from the experimental tank and separate them from naive fish.

Configuração de Tanque Experimental e Configuração de Gravação de Vídeo para Testes Comportamentais em Zebrafish Adulto

Analysis of Recorded Videos for Behavioral Testing of Adult Zebrafish

Open the Analysis Software for examining fish behavioral test data. To elaborate on a new template, click on New from template, then Apply a pre-defined template followed by From video file. Then select a video from the template set up.In the Parameters window, select the model fish to adult zebrafish, the arena to Open field, square to one arena and the number of subjects per arena. Then choose the type of detection by Center point and adjust the frame rate to 30 FPS. Name the experiment as a template and store it in the same folder as the rest of the videos.Under Experiment Settings, review the configured setup. In Arena Settings, right click on the center of the screen and select Grab. From file in the display, select a good quality video image and click Accept to capture this image for the background settings.Using the 19 centimeters width of the tank as a scale, calibrate the captured image to create a calibrated rule. Then draw the arena, and using the frame function, delineate the shape zones. For the novel tank test and shoaling test, draw two equal horizontal boxes on the tank.Draw three equal vertical boxes for the social preference test. Under Trial Control Settings, put an infinite time condition and remove the other components. Next, under Detection Settings, select the video for analysis from the video file and check the detection quality.Click on Auto Detect to adjust the detection and refocus the animal. Under Trial Settings, input one trial. Then under Data Profiles, create results dialogue windows.And under Analyze Profiles, select the parameters to analyze. To copy and use the template, open the template file with behavior analysis software. Then go to File and click Save As to create a new identical file.Alternatively, select New from Template, then apply to custom template. Next, go to Arena Settings to readjust the template. Under Acquisition, select DDS, then click Ready to start to process the video.Once the acquisition is complete, go to track editor to check the processed video. Finally, in Statistics, click on Calculate, then Export Data. Anxiety behavior assessed in a novel tank test of zebrafish exposed to methamphetamine exhibited a decrease in distance traveled, exploration distance and time, and number of visits to the upper zone of the tank.The latency time before the first visit to the upper zone increased significantly. Freezing behavior increased also significantly after three hours of methamphetamine exposure. Social behavior assessed in the shoaling test of the methamphetamine treated fish displayed a behavioral phenotype of social isolation with an increased average and farthest distances between individuals.Social behavior assessed in the social preference test of the methamphetamine treated fish showed a significant decrease in time spent and distance traveled in the conspecific zone.