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 commerc...

<ol>
	<li>Gitler, A. D., Dhillon, P., Shorter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+disease:+models,+mechanisms,+and+a+new+hope.">Neurodegenerative disease: models, mechanisms, and a new hope.</a> Disease Models &amp; Mechanisms. 10, 499-502 (2017).</li><li>Perry, R. J., Watson, P., Hodges, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+and+staging+of+attention+dysfunction+in+early+(minimal+and+mild)+Alzheimer's+disease:+relationship+to+episodic+and+semantic+memory+impairment.">The nature and staging of attention dysfunction in early (minimal and mild) Alzheimer's disease: relationship to episodic and semantic memory impairment.</a> Neuropsychologia. 38, 252-271 (2000).</li><li>Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+in+zebrafish+(Danio+rerio).">Learning and memory in zebrafish (Danio rerio).</a> Methods in Cell Biology. 134, (2016).</li><li>Davidson, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+a+high-energy+diet+on+hippocampal-dependent+discrimination+performance+and+blood-brain+barrier+integrity+differ+for+diet-induced+obese+and+diet-resistant+rats.">The effects of a high-energy diet on hippocampal-dependent discrimination performance and blood-brain barrier integrity differ for diet-induced obese and diet-resistant rats.</a> Physiology and Behavior. 107, 26-33 (2012).</li><li>Yang, M., Silverman, J. L., Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+three-chambered+social+approach+task+for+mice.">Automated three-chambered social approach task for mice.</a> Current Protocols in Neuroscience. 56 (1), (2011).</li><li>Remmelink, E., Smit, A. B., Verhage, M., Loos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+discrimination-+and+reversal+learning+in+mouse+models+within+4+days+and+without+prior+food+deprivation.">Measuring discrimination- and reversal learning in mouse models within 4 days and without prior food deprivation.</a> Learning and Memory. 23, 660-667 (2016).</li><li>Salas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropsychology+of+learning+and+memory+in+teleost+fish.">Neuropsychology of learning and memory in teleost fish.</a> Zebrafish. 3, 157-171 (2006).</li><li>Kalueff, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+comprehensive+catalog+of+zebrafish+behavior+1.0+and+beyond.">Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.</a> Zebrafish. 10, 70-86 (2013).</li><li>Luchiaria, A. C., Salajanb, D. C., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+alcohol+administration:+Effects+on+performance+of+zebrafish+in+a+latent+learning+task.">Acute and chronic alcohol administration: Effects on performance of zebrafish in a latent learning task.</a> Behavior Brain Research. 282, 76-83 (2015).</li><li>Fadool, J., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+model+system+for+the+study+of+eye+genetics.">Zebrafish: A model system for the study of eye genetics.</a> Progress in Retinal and Eye Research. 27, 89-110 (2008).</li><li>Fernandes, Y. M., Rampersad, M., Luchiari, A. C., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+learning+in+the+multichamber+tank:+A+new+learning+paradigm+for+zebrafish.">Associative learning in the multichamber tank: A new learning paradigm for zebrafish.</a> Behavioural Brain Research. 312, 279-284 (2016).</li><li>Reider, M., Connaughton, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+exposure+to+methimazole+increases+anxiety+behavior+in+zebrafish.">Developmental exposure to methimazole increases anxiety behavior in zebrafish.</a> Behavioral Neuroscience. , (2015).</li><li>Capiotti, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycemia+induces+memory+impairment+linked+to+increased+acetylcholinesterase+activity+in+zebrafish+(Danio+rerio).">Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).</a> Behavioural Brain Research. 274, 319-325 (2014).</li><li>May, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Object+recognition+memory+in+zebrafish.">Object recognition memory in zebrafish.</a> Behavioural Brain Research. 296, 199-210 (2016).</li><li>Mathur, P., Lau, B., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+place+preference+behavior+in+zebrafish.">Conditioned place preference behavior in zebrafish.</a> Nature Protocols. 6, 338-345 (2011).</li><li>Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+genes+to+brain,+behavior+and+neurological+diseases:+What+can+we+learn+from+zebrafish.">Linking genes to brain, behavior and neurological diseases: What can we learn from zebrafish.</a> Genes, Brain and Behavior. 3, 63-74 (2004).</li><li>Kily, L. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+a+zebrafish+model+of+drug+dependency+suggest+conservation+of+neuro-adaptation+pathways.">Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways.</a> Journal of Experimental Biology. 211, 1623-1634 (2008).</li><li>Webb, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+reward+mutants+reveal+novel+transcripts+mediating+the+behavioral+effects+of+amphetamine.">Zebrafish reward mutants reveal novel transcripts mediating the behavioral effects of amphetamine.</a> Genome Biology. 10, (2009).</li><li>Clayman, C. L., Malloy, E. J., Kearns, D. N., Connaughton, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+behavioral+effects+of+ethanol+pre-exposure+in+male+and+female+zebrafish+(Danio+rerio).">Differential behavioral effects of ethanol pre-exposure in male and female zebrafish (Danio rerio).</a> Behavioural Brain Research. 335, 174-184 (2017).</li><li>Ruhl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+administration+of+THC+impairs+spatial+but+not+associative+memory+function+in+zebrafish.">Acute administration of THC impairs spatial but not associative memory function in zebrafish.</a> Psychopharmacology. 231, 3829-3842 (2014).</li><li>Gellermann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chance+orders+of+alternating+stimuli+in+visual+discrimination+experiments.">Chance orders of alternating stimuli in visual discrimination experiments.</a> The Pedagogical Seminary and Journal of Genetic Psychology. 42, 206-208 (1933).</li><li>Gleeson, M., Connaughton, V., Arneson, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+hyperglycaemia+in+zebrafish+(Danio+rerio)+leads+to+morphological+changes+in+the+retina.">Induction of hyperglycaemia in zebrafish (Danio rerio) leads to morphological changes in the retina.</a> Acta Diabetologica. 44, 157-163 (2007).</li><li>Connaughton, V. P., Baker, C., Fonde, L., Gerardi, E., Slack, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+immersion+in+an+external+glucose+solution+differentially+affects+blood+sugar+values+in+older+versus+younger+zebrafish+adults.">Alternate immersion in an external glucose solution differentially affects blood sugar values in older versus younger zebrafish adults.</a> Zebrafish. 13, 87-94 (2016).</li><li>Goldsmith, J. R., Jobin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Think+small:+Zebrafish+as+a+model+system+of+human+pathology.">Think small: Zebrafish as a model system of human pathology.</a> Journal of Biomedicine and Biotechnology. 2012, 817341 (2012).</li><li>Kalueff, A. V., Stewart, A. M., Gerlai, R., Court, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+an+emerging+model+for+studying+complex+brain+disorders.">Zebrafish as an emerging model for studying complex brain disorders.</a> Trends in Pharmacological Sciences. 35, 63-75 (2014).</li><li>Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+learning+in+zebrafish+(Danio+rerio).">Associative learning in zebrafish (Danio rerio).</a> Methods in cell biology. 101, 249-270 (2011).</li></ol>

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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			Recommend two of different colors; one for choice latency and one for time to completion</td>
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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...

Watch this Scientific Journal Video about The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System at JoVE.com

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

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 ...

We show that a three-chamber choice task developed for rodents can be adapted to assess the acquisition and reversal of visual discrimination learning in zebrafish. Our behavioral chamber is designed to assess cognitive performance. Once acquired, zebrafish remember the task eight weeks later, making it suitable for studies with zebrafish mutants and/or following drug exposure.Demonstrating the procedure will be Dr.Cassie Rowe, a formal doctoral student from the Connaughton Laboratory. To prepare a three-chamber choice testing chamber, fix an aluminum U-shaped channel to the interior glass walls of a 40 liter 50 by 30 by 30 cubic centimeter aquarium to separate the aquarium into three chambers with the central chamber measuring 10 by 30 by 30 cubic central centimeters. Then fit 10 centimeter high opaque dividers constructed out of gray PVC sheets into both sides of the aluminum channels.For each discrimination task, use hook and loop tape to place individual beige, black, or white felt pieces onto the outer back, side, and bottom of the choice chambers. As a reward, place four adult zebrafish that otherwise will not be used in the study in a small clear tank in the far back corner of each choice chamber to create a group of conspecifics. For group acclimation for each of the three days, attach the beige background to the outside of both choice compartments and submerge a live shoal tank in each of the compartments, then add five to six zebrafish to the central starting chamber with both sliding doors open and allow the fish to roam freely for 30 minutes.The experimental zebrafish should be able to interact and socialize with the shoal fish through the tank as a reward after crossing into either choice compartment during acclamation. A fish is considered to have entered one of the side chambers when its entire body enters the chamber. For individual acclimation, after setting up the chamber in the same manner, place an individual zebrafish in the center starting chamber with the sliding doors closed.After two minutes, open both doors simultaneously and make sure that the first swims from the central chamber through either door a total of 10 times in 30 minutes. Reward the fish each time it enters one of the side chambers. After completing the acclimation, place a white felt piece to the outside of one choice compartment and a black felt piece to the outside of the other choice compartment and place a shoal reward only in one of the choice compartments.This becomes the rewarded side. To begin the acquisition, place a single experimental fish in the starting chamber with the choice compartments closed off. After a two-minute acclimation, simultaneously open both doors and start a stopwatch to assess the choice latency.To denote the choice response, when the fish makes a choice by entering one of the side compartments, stop the timer. If the fish correctly selects the preferred side, immediately close the door between the central chamber in that side to restrict the fish to the preferred side for one minute to allow the first to be rewarded by interacting with the shoal tank. Score this trial as C for correct.If the fish swims through the incorrect door, use a net to gently transfer the fish back to the central chamber, close both doors and score the trial as I for incorrect. If the fish does not make a decision within two minutes of the doors being opened, move the fish to the correct side for one minute and score the trial as M for marked. Once the fish has been returned to the central chamber, wait one minute before performing the task again.After each fish has completed eight acquisition trials, categorize fish that have successfully selected the correct side of the tank at least six out of eight times as high performing and the fish that do not meet this criterion as low performing. Once identified, house the high and low-performing fish separately. When the fish have demonstrated the ability to solve a simple discrimination task, attach the black and white felt to the same sides of the choice chambers as for the acquisition task and submerge the shoal tank in the far back corner of the side opposite to that of the previously rewarded choice chamber.Test the fish individually as demonstrated. As the rewarded side is reversed compared to the acquisition task, the reversal task assesses whether the fish can learn where the reward is located irrespective of the color of the background. When a total of eight trials have been completed for the three consecutive treatment days, report the results for the experiments as group averages for each two-trial block on each of three reversal days, keeping the data for high and low-performing fish separate to determine whether the two groups display the same level of performance during reversal as they did during acquisition.After three days of training, experimental animals conditioned using a shoal-based reward take an average of 125 seconds to reach their first decision and an average of 725 seconds to complete the entire individual acclimation task. No significant side preference during acclimation is observed and the number of excluded fish is minimal compared to other previously tested reward types. During the acquisition phase, the number of high-performing animals increases daily with more high-performing fish observed on acquisition day three compared to day one.The initial choice latency for all of the fish decreases across the three acquisition days indicating an improved performance with each day of acquisition. The same trend is observed when only the high-performing fish group are considered. As shown in this discrimination ratio analysis, all of the fish perform above chance by the end of acquisition, indicating that the fish have learned the discrimination task.Following the acquisition of discrimination learning, the fish demonstrate strong reversal behavior and an increased discrimination over the three-day learning period. The three-chamber choice paradigm can also be applied to the examination of disease complications. For example, in this analysis, hyperglycemic zebrafish acclimation and acquisition were evaluated.This task provides a robust assay that can be applied to various studies examining behaviorally-linked diseases such as hyperglycemic complications of diabetes, Alzheimer's and dementia.

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3.6K Views.  American University. We show that a three-chamber choice task developed for rodents can be adapted to assess the acquisition and reversal of visual discrimination learning in zebrafish. Our behavioral chamber is designed to assess cognitive performance. Once acquired, zebrafish remember the task eight weeks later, making it suitable for studies with zebrafish mutants and/or following drug exposure.Demonstrating the procedure will be Dr.Cassie Rowe, a formal doctoral student from the Connaughton Laboratory....