Name: The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System (Video) | JoVE Journal
Uploaded: 2021-04-14T15:00:00.000+00:00
Duration: 7 min 55 s
Description: Learn the three-chamber choice task for assessing visual discrimination learning and long-term memory in zebrafish. This method enables cognitive studies with mutants and drug exposure models.

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5 minutes

We thank Sabrina Jones for her assistance adapting a rodent three-chamber choice paradigm to the zebrafish model and Jeremy Popowitz and Allison Murk for their help on behavior collection days, assistance with running trials, animal care, and tank set-up. Special thanks also to James M. Forbes (Mechanical Engineer) for his assistance with the 3-chamber choice tank design and construction.
Funding: VPC and TLD received a joint Faculty Research Support grant (FRSG) from American University College of Arts and Sciences. CJR received support from American University College of Arts and Sciences Graduate Student Support.

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at American University (protocol # 1606, 19-02).
1. Animals
<ol>
	<li>Animal rearing and maintenance
	<ol>
		<li>Obtain adult wild-type zebrafish (Danio rerio) aged 4&#8211;11 months as embryos and rear them in-house.</li>
		<li>Maintain the fish in an aquatic rack system at 28&#8211;29 &#176;C on a 14-h light:10-h dark photoperiod.</li>
		<li>Feed the fish twice per day with commercial flakes and supplement with live Artemia.</li>
		<li>Choose fish randomly from these stock tanks for behavioral experiments.</li>
	</ol>
	</li>
	<li>Upon experiment completion, anesthetize the animals by immersion in 0.02% tricaine for 2 min or until there is a lack of motor coordination and reduced respiration rate for later molecular and/or neurochemical analyses.</li>
</ol>
2. Three-chamber choice testing chamber
NOTE: This behavioral technique was modified from Ruhl et al.20.
<ol>
	<li>Chamber construction
	<ol>
		<li>Modify the behavioral chamber26&#8212;a 40 L aquarium (50 x 30 x 30 cm3)&#8212; to have a central or starting chamber (10 x 30 x 30 cm3), separated from two side choice chambers (each 20 x 30 x 30 cm3, Figure 1A).</li>
		<li>Construct the three compartments using an aluminum &#8220;U-shaped&#8221; channel affixed to the interior glass walls with aquarium sealant, to separate the tank into three chambers.</li>
		<li>Construct opaque dividers, 10 cm high, out of grey PVC sheet fit into the aluminum channels on either side. Make each divider out of 2 pieces, each equal in size: a stationary bottom piece permanently mounted within the tank and a moveable top piece that moves up and down in the aluminum tracks.</li>
		<li>Glue extra-large binder clips to the top of the grey PVC sheets to act as handles.</li>
		<li>Using a permanent marker, draw small horizontal lines 10 cm above the adhered grey PVC pipe on the outside of the tank. 
		NOTE: This mark is the point to which the top grey PVC sheet will be opened to allow access to either side.</li>
		<li>Add control (system) water to the tank to a level of 25 cm from bottom to top of the tank, or ~ 30 L. Place glass aquarium heaters into each section of the chamber for 24 h prior to testing to bring the temperature to 28.5 &#176;C. 
		NOTE: Remove the heaters at the start of the behavior session and perform a full water exchange after two days of use.</li>
	</ol>
	</li>
	<li>Discrimination setup
	<ol>
		<li>For each discrimination task, individually place colored felt pieces (beige, black, or white) on the outer back, side, and bottom of the choice chambers using Velcro (Figure 1B&#8211;D). 
		NOTE: The central chamber should have no background color associated with it.</li>
	</ol>
	</li>
	<li>As a reward, create a group of conspecifics (shoal) by placing 4 adult zebrafish, who will not otherwise be used in the study, in a small, clear tank in the far back corner of each choice chamber (Figure 1B&#8211;D). 
	NOTE: Choose shoal fish randomly from stock tanks each day, with at least one male and one female of the same age and size as the experimental fish in each shoal tank.</li>
</ol>
3. Behavioral tasks
<ol>
	<li>Acclimation 
	NOTE: Acclimation to the behavior chamber consists of three days of training; two days of group acclimation followed by one day of individual training.</li>
	<li>Group acclimation
	<ol>
		<li>Attach the beige (neutral) felt background to the outside of both choice compartments, and submerge a live shoal tank in each of the choice compartments (Figure 1B).</li>
		<li>Add five-six zebrafish to the center starting chamber with both sliding doors open, and allow the fish to roam freely for 30 min. 
		NOTE: The experimental zebrafish should be able to interact and socialize with these shoal fish through the tank as a reward after crossing into either choice compartment during acclimation. A fish is considered to have entered one of the side chambers when its entire body enters the chamber.</li>
		<li>Repeat this procedure with the same experimental fish for a second day (2 days of group acclimation). 
		NOTE: Do not keep the same groups of fish.</li>
	</ol>
	</li>
	<li>Individual acclimation
	<ol>
		<li>Chamber setup: Attach the beige (neutral) felt background to the outside of both choice compartments, and submerge a live shoal tank in both of the choice compartments, as in group acclimation (Figure 1B,E).
		<ol>
			<li>Place an individual zebrafish in the central starting chamber for 2 min with the sliding doors closed, and after the 2-min period, open both doors simultaneously.</li>
			<li>Make sure that each fish swims from the central chamber through a door for a total of 10 times, regardless of which side. Reward the fish each time it enters one of the side chambers (1 day of individual acclimation). 
			NOTE: If a fish is unable to complete this task 10 times within a 30-min period or refuses to leave the starting chamber at all, exclude it from the study.</li>
		</ol>
		</li>
		<li>Data acquisition: Record the number of times the fish swims into either side and the total time it takes to complete the task.</li>
	</ol>
	</li>
	<li>Acquisition 
	NOTE: After acclimation, zebrafish began a 3-day acquisition task.
	<ol>
		<li>Chamber setup: Attach a white felt piece to the outside of one choice compartment and a black felt piece to the outside of the other choice compartment (Figure 1C,F). 
		NOTE: Alternate the background color of each side daily using a pseudorandom schedule37.
		<ol>
			<li>For the duration of this phase of training, place a shoal-reward only placed in one of the choice compartments; this becomes the rewarded side.</li>
			<li>To begin acquisition, place a single experimental fish in the starting chamber for a 2-min period with the choice compartments closed off.</li>
			<li>After the 2-min acclimation, simultaneously open both doors, giving access to both choice compartments, and start the stopwatch to assess the choice latency.</li>
			<li>Using a biased design, randomly assign the fish either a black or white preference (i.e., either W+/B- or B+/W-), meaning that the shoal is placed in either the black (B+) or white (W+) choice compartment.</li>
		</ol>
		</li>
		<li>Denoting the choice response
		<ol>
			<li>Once the fish makes a choice by entering one of the side compartments, stop the timer.</li>
			<li>If the fish correctly chooses the preferred side, immediately close the door between the central chamber and that side to restrict the fish to the preferred side for 1 min, and allow it to be rewarded by interacting with the shoal tank (Figure 1C,F). Score this trial as &#8220;C&#8221; for &#8220;Correct&#8221; (rewarded).</li>
			<li>If the fish swims through the incorrect door, transfer it back to the central chamber, close both doors, and score the trial as &#8220;I&#8221; for &#8220;incorrect&#8221; (non-rewarded).</li>
			<li>If the fish does not make a decision within 2 min after the doors are opened, move the fish to the correct side for 1 min, and score the trial as &#8220;M&#8221; for &#8220;marked&#8221; (force-rewarded).</li>
			<li>When transferring/moving fish back into the starting chamber, gently guide the fish into the central chamber using a fish net as a shepherding tool. 
			NOTE: Do not scoop the fish out of the water and replace it into the starting chamber as this could affect the behavioral assay.</li>
			<li>Once the fish returns to the central chamber, wait for 1 min before performing the task again. Ensure that each fish performs the task 8 times.</li>
		</ol>
		</li>
		<li>Data acquisition
		<ol>
			<li>For each experimental fish, record the time to first decision (or choice latency) and the individual scores (C, I, or M) for each of the 8 acquisition trials (section 3.4.2) in order.</li>
			<li>Report the results for these experiments were reported as group averages for each trial on each acquisition day.</li>
			<li>Once a fish has completed a trial, categorize it as either a &#8220;high performing&#8221; fish or a &#8220;low performing&#8221; fish. 
			NOTE: A fish was considered &#8220;high performing&#8221; if it successful chose the correct side of the tank in at least 6 of the 8 total trials for the day. Any fish that does not meet this criterion is a &#8220;low performer&#8221;.</li>
			<li>Once identified, house high performing and low performing fish separately.</li>
			<li>Categorize the fish as &#8220;high&#8221; or &#8220;low&#8221; performers on each of the three days of acquisition, after a fish has completed the trials. 
			NOTE: At the end of the third day of acquisition, fish remain as either &#8220;high&#8221; or &#8220;low&#8221; performers for the duration of the study. 
			NOTE: Some fish that were initially in the &#8216;low performing&#8217; group learn the task on acquisition day 2 or 3. When this happens, the initial &#8216;low performing&#8217; fish may be moved into the &#8216;high performing&#8217; group. Do not move the fish between groups in this manner after day 3 (the end of acquisition).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Experimental treatment
<ol>
	<li>After the acquisition period, when the fish demonstrate the ability to solve a simple discrimination task between the black and white background, start the treatment regimen for the experimental zebrafish. 
	NOTE: To show the applicability of this method, this study shows two experimental designs:
	<ol>
		<li>Longitudinal study
		<ol>
			<li>Return the experimental fish to their holding tanks for 8 weeks. Maintain the fish in standard tanks with daily water changes, and feed them twice daily. 
			NOTE: Do not conduct any behavioral training during these 8 weeks in the holding tanks.</li>
			<li>Perform reversal assessment after this period to assess whether the zebrafish can solve the reversal task after 8 weeks without training.</li>
		</ol>
		</li>
		<li>Hyperglycemia: Expose the experimental groups to water (handling stress control), mannitol (1%&#8211;3%, osmotic control), or glucose (1%&#8211;3%) for 4 or 8 weeks22,23. 
		NOTE: Do not conduct any behavioral training during these 4 or 8 weeks.</li>
	</ol>
	</li>
</ol>
5. Reversal
NOTE: Following experimental manipulation (as in section 4.2), the fish are tested in the final part of the 3-chamber choice paradigm&#8212;reversal. To do this, the rewarded side is reversed (compared to acquisition) such that fish previously rewarded with a shoal on the white side are now rewarded with a shoal on the black side and vice versa. In this way, reversal assesses whether the fish have learned where the reward (shoal) is located, irrespective of the color of the background.
<ol>
	<li>Chamber setup
	<ol>
		<li>Attach black felt to the outside of one of the choice chambers and white felt to the outside of the other, making sure that the black and white sides are the same sides as in the acquisition trials (section 3.4).</li>
		<li>Submerge the shoal tank in the far back corner of the side that is the opposite of the previously rewarded choice chamber (Figure 1D,G). 
		NOTE: In other words, fish previously rewarded on the white side are now rewarded on the black side and vice versa.</li>
		<li>Test the fish individually as in section 3.5. Begin by placing a single experimental fish in the starting chamber for a 2-min period, and close off access to the choice compartments.</li>
		<li>Simultaneously open both sides of the chamber. 
		NOTE: Complete a total of 8 trials each day for three consecutive treatment days.</li>
	</ol>
	</li>
	<li>Denoting the choice response
	<ol>
		<li>If the fish correctly chooses the preferred color, immediately close the door to the central chamber for 1 min, allowing the fish to interact with the shoal reward. Score this trial as &#8220;C&#8221; for &#8220;Correct&#8221; (rewarded).</li>
		<li>If the fish swims through the incorrect door, transfer it back into the central chamber, close both doors, and score this trial as &#8220;I&#8221; for &#8220;incorrect&#8221; (non-rewarded).</li>
		<li>If the fish does not make a decision within 2 min after the doors are opened, move the fish to the correct side, and score the trial as &#8220;M&#8221; for &#8220;marked&#8221; (force-rewarded).</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>For each experimental zebrafish, record the choice latency and the individual scores (C, I, M), in order, for each trial.</li>
		<li>Report the results for these experiments as group averages for each two-trial block on each of the 3 reversal days.</li>
		<li>Keep the data for high and low performing fish separate to determine whether they display the same level of performance during reversal as they did during acquisition.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

Although there has been tremendous growth in the amount and variety of neuroscience research performed using zebrafish in the past 15 years24, behavioral assays are lacking in this species compared to mammalian model systems11,25,26. Here, we show that a three-chamber choice task developed for use with rodents can be adapted to assess the acquisition and reversal of a visual discrimination learning in zeb...

Neurodegenerative diseases are age-dependent, debilitating, and incurable. These diseases are increasing in prevalence, resulting in an urgent need to improve upon and develop new therapeutic strategies. The onset and presentation of each disease is unique, as some affect language, motor, and autonomic brain regions, while others cause learning deficits and memory loss1. Most notably, cognitive deficits and/or impairment are the most prevalent complications across all neurodegenerative diseases2. In hopes of shedding light on the underlying mechanisms involved in these neurodegenerative diseases, the use of many different model systems (including single-celled organisms to Drosophila to higher-order vertebrates such as rodents and humans) have been employed; however, the majority of neurodegenerative diseases remain incurable.
Learning and memory are highly conserved processes among organisms as constant changes to the environment require adaptation3. Impairment in both cognition and synaptic plasticity has been demonstrated in several rodent models. Specifically, well-established behavioral assays use associative learning to assess cognitive changes following various impairment-induced diseases and disorders4. Additionally, contrast discrimination reversal assesses cognitive deficits because it involves higher-order learning and memory functions, and reversal depends on inhibition of a previously learned association. The widely used three-chamber choice task elucidates possible deficits in learning and memory pathways of the central nervous system5,6. Recently, this field has expanded to include non-mammalian models, such as zebrafish (Danio rerio), as several paradigms have been developed for a range of ages from larvae to adults7,8.
Zebrafish provide a balance of complexity and simplicity that is advantageous for the assessment of cognitive impairments with behavioral techniques. First, zebrafish are amenable to high-throughput behavioral screening given their small size and prolific reproductive nature. Second, zebrafish possess a structure, the lateral pallium, which is analogous to the mammalian hippocampus as it has similar neuronal markers and cell types7. Zebrafish are also able to acquire and remember spatial information9 and, like humans, are diurnal10. Therefore, it is not surprising that zebrafish are being used as a model for neurodegenerative diseases with increasing frequency. However, the absence of appropriate behavioral assays has made it difficult to apply the zebrafish model for cognitive assessments. Published work using zebrafish-specific behavioral assays include associative learning tasks11, anxiety behavior12, memory13, object recognition14, and conditioned-place-preference15,16,17,18,19. Though there have been many developments with respect to zebrafish behavioral assays, counterparts for some tests of cognitive functions in rodents have yet to be developed for use with zebrafish18.
Building on previous studies from our lab, we modeled/developed a cognitive task in zebrafish based on the three-chamber choice task used with rodents using social interaction as a reward. Additionally, we expanded upon the associative learning aspect of the behavioral task and incorporated contrast discrimination reversal in hopes of further developing this behavioral task to assess cognitive impairment. This enabled us to examine both the initial acquisition of discrimination learning and the subsequent inhibition of that learning in the reversal phase. In the current study, we demonstrate that this procedure provided a reliable method for assessing cognitive functioning in zebrafish following glucose immersion for 4 or 8 weeks.

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the three-chamber choice behavioral task using zebrafish as a model system

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

Acclimation to the behavior chamber involves three days of training: 2 days of group acclimation followed by 1 day of individual acclimation. However, because we could not distinguish individual zebrafish from one another, we were only able to collect data during individual acclimation. At this time, experimental animals (n = 30), conditioned using a shoal-based reward, took an average of 125.11 s to reach their first decision (Figure 2A) and an average of 725.34 s (12 min) to complete the e...

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

the-three-chamber-choice-behavioral-task-using-zebrafish-as-model

Research

JoVE Journal

Behavior

The video describes the avoidance and thigmotaxis assay of zebrafish larvae. The video examines the behavior of zebrafish larvae in response to visual aversive stimulus.

Zebrafish Avoidance and Thigmotaxis Assay: A High-Throughput Method to Examine Larval Behavior in Response to Aversive Visual Stimulus

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

A novel method for reducing variability when exposing fish to drugs is explained.  Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.

Behavioral Approaches to Studying Innate Stress in Zebrafish

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

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.

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

Multi-chamber social preference tasks have been adapted from rodents to zebrafish, facilitating the study of social behavior in model organisms. This protocol validates a low-cost 3-chamber open-tank task using webcams and open-source software to measure zebrafish social preference, making it accessible to undergraduate research and teaching.

Low-Cost Alternative for Examining Social Preference in Zebrafish (Danio rerio) Using the 3-Chamber Open-Tank Free-Swim Task

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

Learn the three-chamber choice task for assessing visual discrimination learning and long-term memory in zebrafish. This method enables cognitive studies with mutants and drug exposure models.

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

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