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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ne....

Protocol

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

  1. Perform all tests (presen.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

This work was supported by "Agencia Estatal de Investigación" 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).

....

Materials

NameCompanyCatalog NumberComments
Aquarium Cube shapeBlau Aquaristic7782025Cubic Panoramic 10  (10 L, 20 cm x 20 cm x 25 cm, 5 mm)
Ethovision softwareNoldusEthovision XTVersion 12.0 or newer
GigE cameraImaging Development SystemsUI-5240CP-NIR-GL
GraphPad Prism 9.02GraphPad software IncGraphPad Prism 9.02 For Windows
IDS camera managerImaging Development Systems
LED backlight illuminationQuirumedGP-G2
SPSS SoftwareIBMIBM SPSS v26
uEye Cockpit software Imaging Development Systemsversion 4.90

References

  1. Raldúa, D., Piña, B. In vivo zebrafish assays for analyzing drug toxicity. Expert Opinion on Drug Metabolism & Toxicology. 10 (5), 685-697 (2014).
  2. Faria, M., Prats, E., Bellot, M., Gomez-Canela, C., Raldúa, D.

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