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

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 FASD^2,3. 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

1. Raise and breed zebrafish as described¹⁶.
2. Ethanol exposure
   1. Choose the appropriate developmental stage at which to conduct the ethanol exposure. In this protocol embryos were exposed to ethanol at 24 hpf.
   2. Place eggs in 1.0% ethanol volume/volume for 2 h¹⁵. A ratio of 1 egg per mL of EM is good practice. Specifically, place embryos in 50 mL of embryo medium (EM; please see Westerfield¹⁶ for the EM recipe) then remove 500 µL of EM and replace with 500 µL of ethanol.
   3. After ethanol exposure, raise embryos as described¹⁷. Assay social behavior when fish are 16 weeks old.

2. Randomization and tank setup

1. Using an online random sequence generator, randomize all trials a priori to conducting the behavioral assays. Ensure that the stimulus side and the treatment groups vary randomly.
2. To avoid any confounding factors such as time of day or day of testing, start and end the behavioral assay at the same time every day and conduct the behavioral testing on consecutive days until all experimental fish have been tested.
3. For this assay (Figure 1), use a 37 L tank (50 cm x 25 cm x 30 cm, L x W x H) with 1.4 L tanks placed outside along the width of the tank as previously described¹⁷.
4. Line the back and the bottom of the 37 L tank with white corrugated plastic to increase the contrast between the experimental fish and the background to improve video tracking.
5. Place the corrugated plastic on the outer wall of the 1.4 L tanks to increase the contrast of the social stimulus for the experimental fish. Finally, place white corrugated plastic between the 1.4 L tanks and 37 L tanks; this opaque barrier is used to prevent the experimental fish from viewing the social stimulus during habituation.
6. Place the camera at a distance that is far enough to capture the entire length of the 37 L tank plus half of the 1.4 L tanks and accurately track the adult experimental fish.
   NOTE: Seeing which side holds the stimulus ensures that researchers have labelled the tracking zones correctly and provides redundancy as a backup.
7. If no infrared tracking is being used make sure to illuminate the 37 L tank. Use any commercially available aquarium hood lights with a 15 W T8 full spectrum lamp.
   ​NOTE: If multiple arenas are being set up use identical aquarium hoods with identical lamps.

3. Conducting the social assay

1. Begin by filling the 37 L tank used for the behavioral assay with water that is identical to the water used in the housing rack. Ensure that the water temperature is within 2 °C of the housing rack.
2. At the end of the day empty the 37 L tank. Begin each testing day with fresh water. Ensure the water level in the 37 L tank and the water level in the 1.4 L tanks are identical. If the temperature of the room does not keep the water in the 37 L tank at 28.5 °C 2 °C, then replace the water with warm water at 28.5 °C.
3. Next set up the zones of interest. Consult user's manual tracking software of choice to create zones. In this protocol along the length of the 37 L tank on the top and the bottom a tape measure was used to mark 5 cm increments. Given that the testing tank is 50 cm, 5 cm increments lead to 10 zones that are 5 cm each.
4. Using the marks made on the 37 L tank as a reference, use the software to construct the zones by connecting the 5 cm point on the top to the corresponding 5 cm point on the bottom. Additionally, create a zone along the bottom and along the stimulus. Customize zones of interest.
5. Use the tracking software in this protocol to measure the distance from a zone and the duration spent in the zone. In this protocol 12 zones were used.
6. Quantify the time spent in zones 1 to 10 and the distance from stimulus and the bottom as Zone 1, as the zone closest to the stimulus while Zone 10 is the furthest away from the stimulus.
7. Next, select the two males and two females that will be used for the social stimulus. Best practice would be to use males and females from the same cohort as the experimental fish. If that is not possible try to find fish that match the strain, age, and size of the experimental fish.
8. Transfer the experimental fish from the housing tank to the testing arena (37 L tank). Use a net to catch the fish in the housing tank. Place the net with the fish in it, into a container with fish water.
   NOTE: Using this approach will reduce the stress on experimental fish while moving between tanks.
9. Place the experimental fish in the center of the testing arena.
10. Once the detection settings are satisfactory based on user's software of choice (see user's manual), begin the 20 min trial. During the first 10 min, leave the opaque barrier between the 37 L tank and 1.4 L tanks in place. This will prevent the experimental fish from seeing the social stimulus, consisting of two male and two female zebrafish, and allow the fish to acclimatize to the testing arena^13,17.
11. After 10 min carefully remove the opaque barriers by pulling them from behind the tank; this will allow the experimental fish to see the social stimulus.
12. Use tracking software to track and quantify the behavior of the experimental fish ^{11, 12, 17} based on the user's preferences and software manual. In the current protocol, quantify the distance from the social stimulus and the bottom of the tank as well as the time spent in all zones.
13. Analyze data using traditional data analysis tools.

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 behavior^5,19. 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...

Materials

Name	Company	Catalog Number	Comments
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