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>

<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>

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.

<table>
	<tbody>
		<tr>
			<td>Champion Sports Stopwatch Timer Set: Waterproof, Handheld Digital Clock Sport Stopwatches with Large Display for Kids or Coach - Bright Colored 6 Pack</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Champion-Sports-910SET-Stopwatch-Timer/dp/B001CD9LJK/ref=sr_1_17?dchild=1&#38;keywords=stopwatch+for+sports&#38;qid=1597081570&#38;sr=8-17 
			Recommend two of different colors; one for choice latency and one for time to completion</td>
		</tr>
		<tr>
			<td>Coofficer Extra Large Binder Clips 2-Inch (24 Pack), Big Paper Clamps for Office Supplies, Black</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Coofficer-Binder-2-Inch-Clamps-Supplies/dp/B07C94YCR5/ref=sr_1_3_sspa?dchild=1&#38;keywords=large+binder+clips&#38;qid=1597081521&#38;sr=8-3-spons&#38;psc=1&#38;spLa=ZW5jcnlwdGVkUXVhbGlmaWVyPUExUENWUTRZVjlIWEVPJmVuY3J5cHRlZElkPUEwNDQ5NDU0MlpSREkwTFlLSThVQiZlbmNyeXB0ZWRBZElkPUEwMTg5NDI3MllRV1EzOUdWTVpSOCZ3aWRnZXROYW1lPXNwX2F0ZiZhY3Rpb249Y2xpY2tSZWRpcmVjdCZkb05vdExvZ0NsaWNrPXRydWU=</td>
		</tr>
		<tr>
			<td>Marineland&#174; Silicone Aquarium Sealant</td>
			<td>Petsmart</td>
			<td>Item #2431002</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC (Polyvinyl Chloride) Sheet, Opaque Gray, Standard Tolerance, UL 94/ASTM D1784, 0.125&#34; Thickness, 12&#34; Width, 24&#34; Length</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Polyvinyl-Chloride-Standard-Tolerance-Thickness/dp/B000MAMGEQ/ref=sr_1_2?dchild=1&#38;keywords=grey+PVC+sheet&#38;qid=1597081440&#38;sr=8-2</td>
		</tr>
		<tr>
			<td>Steelworks&#160;1/4-in W x 8-ft L Mill Finished Aluminum Weldable Trim U-shaped Channel</td>
			<td>Lowes</td>
			<td>Item #55979Model #11377</td>
			<td>https://www.lowes.com/pd/Steelworks-1-4-in-W-x-8-ft-L-Mill-Finished-Aluminum-Weldable-Trim-Channel/3058181</td>
		</tr>
		<tr>
			<td>Tetra 10 Gallon Fish tank</td>
			<td>Petsmart</td>
			<td>Item #5271256</td>
			<td></td>
		</tr>
		<tr>
			<td>Top Fin Fine Mesh Fish Net (3 in)</td>
			<td>Petsmart</td>
			<td>Item #5175115</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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

Research

JoVE Journal

Behavior

3.7K Views.  American University. 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.

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

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

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

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 rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, modifications to the standard task, can be used to systematically tax the neural systems controlling either attention or impulse control. Importantly, these challenges have consistent effects on behavior across laboratories in intact animals and can reveal either enhancements or deficits in cognitive function that are not apparent when rats are only tested on the standard task. The variety of behavioral measures that are collected can be used to determine if other factors (i.e., sedation, motivation deficits, locomotor impairments) are contributing to changes in performance. The versatility of the 5CSRTT is further enhanced because it is amenable to combination with pharmacological, molecular, and genetic techniques.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, which are modifications of the standard task, increase flexibility of the task and can be combined with other manipulations to more fully characterize behavior.

This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. ...

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many neuropsychiatric and neurodevelopmental disorders, including depression, post-traumatic stress disorder, schizophrenia and autism spectrum disorder. Deficits in cognitive function also represent the most difficult symptom domain&#160;to successfully treat, as serotonin reuptake inhibitors and tricyclic antidepressants have only modest effects. Functional neuroimaging studies and postmortem analysis of human brain tissue implicate the PFC as being a primary region of dysregulation in patients with these disorders. However, preclinical behavioral assays used to assess these deficits in mouse models which can be readily manipulated genetically and could provide the basis for studies of new treatment avenues have been underutilized. Here we describe the adaptation of a behavioral assay, the attentional set shifting task (AST), to be performed in mice to assess prefrontal cortex mediated cognitive deficits. The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents, providing excellent face, construct and predictive validity.

The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many ...

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Errors as a Means of Reducing Impulsive Food Choice

Nowadays, the increasing incidence of eating disorders due to poor self-control has given rise to increased obesity and other chronic weight problems, and ultimately, to reduced life expectancy. The capacity to refrain from automatic responses is usually high in situations in which making errors is highly likely. The protocol described here aims at reducing imprudent preference in women during hypothetical intertemporal choices about appetitive food by associating it with errors. First, participants undergo an error task where two different edible stimuli are associated with two different error likelihoods (high and low). Second, they make intertemporal choices about the two edible stimuli, separately. As a result, this method decreases the discount rate for future amounts of the edible reward that cued higher error likelihood, selectively. This effect is under the influence of the self-reported hunger level. The present protocol demonstrates that errors, well known as motivationally salient events, can induce the recruitment of cognitive control, thus being ultimately useful in reducing impatient choices for edible commodities.

Giving in to temptation of tasty food may result in long-term overweight problems. This protocol describes how to reduce imprudent preference for edible commodities during hypothetical intertemporal choices in women by associating them with errors.

Nowadays, the increasing incidence of eating disorders due to poor self-control has given rise to increased obesity and other chronic weight problems, and ...

The Emotional Stroop Task: Assessing Cognitive Performance under Exposure to Emotional Content

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The difference shows that people are affected by the emotional content conveyed by the carrier words even though they are irrelevant to the color-naming task at hand. The ESE has been widely deployed with patient populations, as well as with non-selected populations, because the emotion words can be selected to match the tested pathology. The ESE is a powerful tool, yet it is vulnerable to various threats to its validity. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them. The most prevalent threat to the validity of existing ESE studies is sustained effects and habituation wrought about by repeated exposure to emotion stimuli. Consequently, the order of exposure to emotion and neutral stimuli is of utmost importance. We show that in the standard design, only one specific order produces the ESE.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than those of neutral words. This report refers to potential sources of confounding and includes a modal experiment that provides the means to control for them.

The emotional Stroop effect (ESE) is the result of longer naming latencies to ink colors of emotion words than to ink colors of neutral words. The ...

A Two-interval Forced-choice Task for Multisensory Comparisons

We provide a procedure for a psychophysics experiment in humans based on a previously described paradigm aimed to characterize the perceptual duration of intervals within the range of milliseconds of visual, acoustic, and audiovisual aperiodic trains of six pulses. In this task, each of the trials consists of two consecutive intramodal intervals where the participants press the upward arrow key to report that the second stimulus lasted longer than the reference, or the downward arrow key to indicate otherwise. The analysis of the behavior results in psychometric functions of the probability of estimating the comparison stimulus to be longer than the reference, as a function of the comparison intervals. In conclusion, we advance a way of implementing standard programming software to create visual, acoustic, and audiovisual stimuli, and to generate a two-interval forced-choice (2IFC) task by delivering stimuli through noise-blocking headphones and a computer&#39;s monitor.

Psychophysics is essential for studying perception phenomena through sensory information. Here we present a protocol to perform a two-interval forced-choice task as implemented in a previous report on human psychophysics where participants estimated the duration of visual, auditory, or audiovisual intervals of aperiodic trains of pulses.

We provide a procedure for a psychophysics experiment in humans based on a previously described paradigm aimed to characterize the perceptual duration of ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.