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

Summary

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.

Abstract

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.

Introduction

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 system^5,6. 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 adults^7,8.

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-preference^{15,16,17,18,19}. 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.

Protocol

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at American University (protocol # 1606, 19-02).

1. Animals

1. Animal rearing and maintenance
   1. Obtain adult wild-type zebrafish (Danio rerio) aged 4–11 months as embryos and rear them in-house.
   2. Maintain the fish in an aquatic rack system at 28–29 °C on a 14-h light:10-h dark photoperiod.
   3. Feed the fish twice per day with commercial flakes and supplement with live Artemia.
   4. Choose fish randomly from these stock tanks for behavioral experiments.
2. 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.

2. Three-chamber choice testing chamber

NOTE: This behavioral technique was modified from Ruhl et al.²⁰.

1. Chamber construction
   1. Modify the behavioral chamber²⁶—a 40 L aquarium (50 x 30 x 30 cm³)— 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).
   2. Construct the three compartments using an aluminum “U-shaped” channel affixed to the interior glass walls with aquarium sealant, to separate the tank into three chambers.
   3. 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.
   4. Glue extra-large binder clips to the top of the grey PVC sheets to act as handles.
   5. 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.
   6. 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 °C.
      NOTE: Remove the heaters at the start of the behavior session and perform a full water exchange after two days of use.
2. Discrimination setup
   1. 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–D).
      NOTE: The central chamber should have no background color associated with it.
3. 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–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.

3. Behavioral tasks

1. 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.
2. Group acclimation
   1. 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).
   2. 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.
   3. 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.
3. Individual acclimation
   1. 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).
      1. 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.
      2. 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.
   2. Data acquisition: Record the number of times the fish swims into either side and the total time it takes to complete the task.
4. Acquisition
   NOTE: After acclimation, zebrafish began a 3-day acquisition task.
   1. 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.
      1. 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.
      2. To begin acquisition, place a single experimental fish in the starting chamber for a 2-min period with the choice compartments closed off.
      3. After the 2-min acclimation, simultaneously open both doors, giving access to both choice compartments, and start the stopwatch to assess the choice latency.
      4. 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.
   2. Denoting the choice response
      1. Once the fish makes a choice by entering one of the side compartments, stop the timer.
      2. 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 “C” for “Correct” (rewarded).
      3. If the fish swims through the incorrect door, transfer it back to the central chamber, close both doors, and score the trial as “I” for “incorrect” (non-rewarded).
      4. 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 “M” for “marked” (force-rewarded).
      5. 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.
      6. 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.
   3. Data acquisition
      1. 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.
      2. Report the results for these experiments were reported as group averages for each trial on each acquisition day.
      3. Once a fish has completed a trial, categorize it as either a “high performing” fish or a “low performing” fish.
         NOTE: A fish was considered “high performing” 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 “low performer”.
      4. Once identified, house high performing and low performing fish separately.
      5. Categorize the fish as “high” or “low” 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 “high” or “low” performers for the duration of the study.
         NOTE: Some fish that were initially in the ‘low performing’ group learn the task on acquisition day 2 or 3. When this happens, the initial ‘low performing’ fish may be moved into the ‘high performing’ group. Do not move the fish between groups in this manner after day 3 (the end of acquisition).

4. Experimental treatment

1. 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:
   1. Longitudinal study
      1. 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.
      2. Perform reversal assessment after this period to assess whether the zebrafish can solve the reversal task after 8 weeks without training.
   2. Hyperglycemia: Expose the experimental groups to water (handling stress control), mannitol (1%–3%, osmotic control), or glucose (1%–3%) for 4 or 8 weeks^22,23.
      NOTE: Do not conduct any behavioral training during these 4 or 8 weeks.

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—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.

1. Chamber setup
   1. 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).
   2. 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.
   3. 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.
   4. Simultaneously open both sides of the chamber.
      NOTE: Complete a total of 8 trials each day for three consecutive treatment days.
2. Denoting the choice response
   1. 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 “C” for “Correct” (rewarded).
   2. If the fish swims through the incorrect door, transfer it back into the central chamber, close both doors, and score this trial as “I” for “incorrect” (non-rewarded).
   3. 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 “M” for “marked” (force-rewarded).
3. Data acquisition
   1. For each experimental zebrafish, record the choice latency and the individual scores (C, I, M), in order, for each trial.
   2. Report the results for these experiments as group averages for each two-trial block on each of the 3 reversal days.
   3. 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.

Results

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...

Discussion

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 systems^11,25,26. 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...

Materials

Name	Company	Catalog Number	Comments
Champion Sports Stopwatch Timer Set: Waterproof, Handheld Digital Clock Sport Stopwatches with Large Display for Kids or Coach - Bright Colored 6 Pack	Amazon	N/A	https://www.amazon.com/Champion-Sports-910SET-Stopwatch-Timer/dp/B001CD9LJK/ref=sr_1_17?dchild=1&keywords=stopwatch+for+sports&qid=1597081570&sr=8-17 Recommend two of different colors; one for choice latency and one for time to completion
Coofficer Extra Large Binder Clips 2-Inch (24 Pack), Big Paper Clamps for Office Supplies, Black	Amazon	N/A	https://www.amazon.com/Coofficer-Binder-2-Inch-Clamps-Supplies/dp/B07C94YCR5/ref=sr_1_3_sspa?dchild=1&keywords=large+binder+clips&qid=1597081521&sr=8-3-spons&psc=1&spLa=ZW5jcnlwdGVkUXVhbGlmaWVyPUExUENWUTRZVjlIWEVPJmVuY3J5cHRlZElkPUEwNDQ5NDU0MlpSREkwTFlLSThVQiZlbmNyeXB0ZWRBZElkPUEwMTg5NDI3MllRV1EzOUdWTVpSOCZ3aWRnZXROYW1lPXNwX2F0ZiZhY3Rpb249Y2xpY2tSZWRpcmVjdCZkb05vdExvZ0NsaWNrPXRydWU=
Marineland® Silicone Aquarium Sealant	Petsmart	Item #2431002
PVC (Polyvinyl Chloride) Sheet, Opaque Gray, Standard Tolerance, UL 94/ASTM D1784, 0.125" Thickness, 12" Width, 24" Length	Amazon	N/A	https://www.amazon.com/Polyvinyl-Chloride-Standard-Tolerance-Thickness/dp/B000MAMGEQ/ref=sr_1_2?dchild=1&keywords=grey+PVC+sheet&qid=1597081440&sr=8-2
Steelworks 1/4-in W x 8-ft L Mill Finished Aluminum Weldable Trim U-shaped Channel	Lowes	Item #55979Model #11377	https://www.lowes.com/pd/Steelworks-1-4-in-W-x-8-ft-L-Mill-Finished-Aluminum-Weldable-Trim-Channel/3058181
Tetra 10 Gallon Fish tank	Petsmart	Item #5271256
Top Fin Fine Mesh Fish Net (3 in)	Petsmart	Item #5175115