Summary

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

Published: February 09, 2024
doi:

Summary

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.

Abstract

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.

Introduction

Prenatal alcohol exposure can lead to a variety of birth defects collectively known as fetal alcohol spectrum disorders (FASD)1. Impaired behavior, such as social difficulties, are common birth defects associated with FASD2,3. Unfortunately, social difficulties frequently result in serious mental health issues4, which can adversely affect the quality of life for individuals with FASD. Thus, understanding the mechanisms responsible for ethanol-induced social defects is paramount.

Zebrafish have biological and behavioral characteristics which make them well suited to advancing our understanding of the mechanisms responsible for ethanol-induced social defects. For instance, zebrafish produce large quantities of transparent externally fertilized eggs; these biological characteristics allow researchers to easily create precise and replicable FASD phenotypes5. To expose embryos to ethanol at 24 h postfertilization (hpf), one simply has to use a dissecting microscope to examine the transparent egg and stage the embryo based on previously published work such as Kimmel et al.6, then place the egg in the desired ethanol concentration for the desired duration. Since the chorion is a weak barrier to alcohol7, the ethanol readily bathes the embryo. To stop the exposure, one simply has to remove the eggs from the ethanol solution. Besides providing researchers with a simple yet accurate method for creating FASD phenotypes, zebrafish also allow researchers to make genetic comparisons to humans because 70% of human genes have a zebrafish orthologue, thus they are a valuable tool for understanding human diseases-related genes8. Additionally, unlike other animal models zebrafish form social groups9 called shoals10. Shoaling behavior can be used to characterize the effects embryonic ethanol exposure has on social behavior11. Furthermore, in zebrafish a social response can be elicited by using computer controlled social stimuli12 or a live social stimulus13.

Previous works have characterized the social response of adult zebrafish in groups14, however a limitation of this approach is the inability to correlate the behavior of an individual fish with a specific measure such as changes in neurotransmitter levels11. The following protocol will give users the ability to characterize the social behavior of an individual adult zebrafish. Since social behavior is acquired for individual fish, users of the protocol can now correlate the acquired behavioral profile of each fish with a dependent outcome. For example, previous work has shown that embryonic ethanol exposure impairs the dopaminergic response to a social stimulus11. While the data shown here has used embryonic ethanol exposure as the independent variable, protocol users can characterize the effects other pharmacological treatments or genetic mutations have on social behavior. Furthermore, protocol users are not limited to examine how embryonic treatments alter behavior but can also determine how acute pharmacological treatments in adult zebrafish impact social behavior15.

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Dakota. 1. Zebrafish housing, care, and embryonic ethanol exposure Raise and breed zebrafish as described16. Ethanol exposure Choose the appropriate developmental stage at which to conduct the ethanol exposure. In this protocol embryos were exposed to ethanol at 24 hpf. Place egg…

Representative Results

Figure 2 has been modified from Fernandes et al.17 and shows that embryonic ethanol exposure blunts the shoaling response by examining the distance from the stimulus. The data in Figure 2 represents the distance from the social stimulus during the 20 min trial. The Y-axis shows the distance in centimeters while the X-axis shows the 20 min trial broken down into 1 min intervals. The black bar along the X-axis represents the time when the o…

Discussion

Zebrafish have a number of biological and behavioral characteristics making them a highly attractive organism for research involving genes, the environment, and behavior5,19. This protocol gives the end user a relatively simple guide to assay social behavior, multiple ways to quantity the social behavior, and has the potential to link the behavioral responses of individual fish with treatments such as embryonic ethanol exposure, genetic mutations, or other pharma…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

Funding to support this research was provided by the National Institutes of Health (NIH)/National Institute on Alcohol Abuse (NIAAA) [R00AA027567] to Y.F.

Materials

1.4-l ZT140 Aquaneering tanks Aquaneering ZT140  Tanks for social stimulus
Aqueon 20" Deluxe Fluorescent Full Hood aquarium light https://www.petco.com/shop/en/petcostore/product/aqueon-aquarium-black-24-fluorescent-deluxe-full-hood-215740 Light for the 37-I tank
Aqueon Standard Open-Glass Glass Aquarium Tank, 10 Gallon https://www.petco.com/shop/en/petcostore/product/aga-10g-20x10x12bk-tank-170917 37-l tank for the social assay
Ethanol  Fisher Scienticfic  BP28184
Ethovision XT tracking system https://www.noldus.com/ethovision-xt
R-Capable Color Basler GigE Camera https://www.noldus.com/ethovision-xt
White corrugated plastic  https://www.homedepot.com/p/Coroplast-48-in-x-96-in-x-0-157-in-4mm-White-Corrugated-Twinwall-Plastic-Sheet-CP4896S/205351385 Plastic to line the back and the bottom of the 37-I tank and back of the tanks used for the social stimulus

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Rampersad, M., Fernandes, Y. Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior. J. Vis. Exp. (204), e65834, doi:10.3791/65834 (2024).

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