The authors would like to thank the Hyde lab members for their thoughtful discussions and the Freimann Life Sciences Center technicians for zebrafish care and husbandry. This work was supported by the Center for Zebrafish Research at the University of Notre Dame, the Center for Stem Cells and Regenerative Medicine at the University of Notre Dame, and grants from National Eye Institute of NIH R01-EY018417 (DRH), the National Science Foundation Graduate Research Fellowship Program (JTH), LTC Neil Hyland Fellowship of Notre Dame (JTH), Sentinels of Freedom Fellowship (JTH), and the Pat Tillman Scholarship (JTH). Figure 1 made with BioRender.com.

Zebrafish were raised and maintained in the Notre Dame Zebrafish facility in the Freimann Life Sciences Center. The methods described in this manuscript were approved by the University of Notre Dame Animal Care and Use Committee (Animal Welfare Assurance Number A3093-01).
1. Shuttle box learning paradigm (Figure 1A)
NOTE: The learning paradigm provides a rapid assessment of cognition regarding associative learning.
<ol>
	<li>Prepare the shuttle box by modifying a 30.5 x 19 x 7.5 cm gel box with a 5 x 19 cm piece of aquarium grade plexiglass added to each side at a 45&#176; angle. Make a line marking the halfway point of the tank to assess when fish have crossed the middle of the tank (Figure 1B).</li>
	<li>Add 800 mL of system water to the shuttle box. Make this water by dissolving 60 mg of Instant Ocean in 1 L of deionized RO water. Fill the water to the middle of the tank to a depth of 5 cm. 
	NOTE: Replace with fresh system water at 28 &#176;C every h or after testing 3 fish.</li>
	<li>Place 2-3 fish into a holding tank containing system water, located in a dark room where the shuttle box assay will be performed.
	<ol>
		<li>In the dark room, place 1 fish in the center of the shuttle box, secure the lid, and attach the electrodes to a power supply. 
		NOTE: The room should remain as dark as possible during acclimation and testing.</li>
	</ol>
	</li>
	<li>Acclimate the fish in the shuttle box for 15 min. 
	NOTE: The investigator should remain in the room during the acclimation period or return to the testing room quietly with ample time before the testing to allow fish to adjust to the investigator&#39;s presence. Successful acclimation can be considered when the fish freely explores the tank.
	<ol>
		<li>If the fish fails to explore, continue acclimation for an additional 15 min. If the fish still fails to acclimate to the shuttle box, remove the fish. Do not use this fish for testing.</li>
	</ol>
	</li>
	<li>Manually shine an 800-lumen red lens flashlight ~2 cm from the gel box wall on the side occupied by the fish, following acclimation. 
	NOTE: Do not start a trial if the fish is resting next to the platinum wire against the wall near the deep ends of the shuttle box.</li>
	<li>Shine the light stimulus directly on the fish and manually follow any lateral movement of the fish with the light to ensure continual visualization of the stimulus (Figure 1C). Continue to provide the light stimulus until either of the following conditions are met.
	<ol>
		<li>Consider the trail successful if the fish crosses over the halfway point of the tank within the 15 s of light exposure. Once the fish crosses the halfway point, stop the light stimulus immediately (Figure 1D).</li>
		<li>Consider the trail as failed if the fish does not cross over the halfway point of the box in 15 s. In this case, use an electrophoresis power supply to apply a negative shock stimulus (20 mV:1 A) alternating 2 s of On, 2 s of Off for a 15 s period (maximum of 4 shocks), or until the fish passes the halfway point of the box, at which point terminate both the light and negative stimulus.</li>
	</ol>
	</li>
	<li>Let the fish rest for 30 s and repeat step(s) 1.5-1.6.2. Keep a detailed record of the order of successful trials (1.6.1) and failed trials (1.6.2). 
	NOTE: Here, we defined learning as the completion of 5 consecutive successful trials. Once the learning has been demonstrated, the fish should be removed from the shuttle box and humanely euthanized.</li>
</ol>
2. Memory paradigm (Figure 1A)
NOTE: This paradigm provides an assessment of the short and long-term memory of a learned associative cognitive task.
<ol>
	<li>Training Period
	<ol>
		<li>Add 800 mL of system water to the shuttle box. Make this water by dissolving 60 mg of Instant Ocean in 1 L of deionized RO water. Fill the water to the middle of the tank to a depth of 5 cm. 
		NOTE: Water should be replaced with fresh system water at 28 &#176;C every h or after testing 3 fish.</li>
		<li>Place 2-3 fish into a holding tank that contains system water, located in a dark room where the shuttle box assay will be performed.</li>
		<li>In the dark room, place 1 fish in the center of the shuttle box, secure the lid, and attach the electrodes to a power supply. 
		NOTE: The room should remain as dark as possible during acclimation and testing.</li>
		<li>Acclimate fish in the shuttle box for 15 min. 
		NOTE: The investigator should remain in the room during acclimation period or return to the testing room quietly with ample time prior to testing to allow fish to adjust to the investigator&#39;s presence. Determine successful acclimation when the fish is freely exploring the tank.</li>
		<li>If the fish fails to explore, continue acclimation for an additional 15 min. If the fish still fails to acclimate to the shuttle box, remove the fish and do not use it for testing.</li>
		<li>After the successful acclimation, manually shine an 800-lumen red lens flashlight ~2 cm from the gel box side wall, on the side of the shuttle box that is occupied by the fish.</li>
		<li>Shine the light stimulus directly on the fish and follow any lateral movement of the fish with the light to ensure continual visualization of the stimulus by the fish.</li>
		<li>While the light is shining on the fish, simultaneously apply the adverse shock stimulus (20 mV:1 A) alternating 2 s On, 2 s Off for 15 s (maximum of 4 shocks), or until the fish passes the halfway point of the box. Once this is achieved, terminate both the light and the adverse stimulus. 
		NOTE: Allow the fish to rest for 30 s then repeat step 2.1.6-2.1.8 for 25 iterations (Figure 1A).</li>
	</ol>
	</li>
	<li>Initial testing
	<ol>
		<li>Allow 15 min of rest to the fish following the training period. Do not remove them from the shuttle box. Test initial memory retention by recording each trial as strictly pass/fail, immediately following this rest period.</li>
		<li>Apply only the light stimulus for up to 15 s and record the responses as follows.
		<ol>
			<li>Consider the trial successful if the fish crosses over the halfway point of the shuttle box within 15 s after starting the light stimulus. Stop the light stimulus immediately when the fish crosses the halfway point.</li>
			<li>Consider the trial as failed if the fish does not cross over the halfway point of the shuttle box 15 s after starting the light stimulus. Stop the light stimulus after 15 s. 
			NOTE: During the initial testing, an adverse stimulus is not applied following a failed attempt.</li>
		</ol>
		</li>
		<li>Repeat step 2.2.2, with a 30 s rest period between trials, and record successful trials (2.2.2.1) and failed trials (2.2.2.2) across 25 trials. This value will serve as an individual reference for each fish.</li>
	</ol>
	</li>
	<li>Immediate memory
	<ol>
		<li>Induce injury immediately following the initial testing period by preferred damage paradigm (e.g., a blunt-force trauma using the modified Marmarou weight drop). House fish individually for an easy identification. Record their initial testing values and return fish to the animal facility. 
		&#8203;NOTE: Fish were injured by blunt-force TBI as previously described22.</li>
		<li>Gather 2-3 undamaged or TBI fish 4 h after initial testing and/or 4 h post-injury (or at the experimental timeframe in question) from the animal facility. Keep all fish in the dark room in individual tanks containing system water.</li>
		<li>Place fish in the center of the shuttle box (prepared with system water as described in 1.1), one fish at a time, and secure the lid. Attach the power supply and allow the fish to acclimate for 15 min.</li>
		<li>Following acclimation, assess immediate memory (strictly pass/fail) by applying only the light stimulus for up to 15 s and record the responses as follows.
		<ol>
			<li>Consider the trial successful if the fish crosses over the halfway point of the box within the 15 s test period. Terminate the light stimulus upon crossing the halfway point.</li>
			<li>Consider the trial as failed if the fish does not cross over the halfway point of the box within 15 s of starting the light stimulus. Terminate the light stimulus after 15 s period is over. 
			NOTE: During this post-injury testing, adverse shock stimulus is not applied following a failed attempt.</li>
		</ol>
		</li>
		<li>Repeat step 2.3.4, with a 30 s rest period between trials, and record the number of successful trials (2.3.4.1) and failed trials (2.3.4.2) across 25 trials.</li>
		<li>Calculate the percent difference in successful trials post-injury to the initial testing period using the equation: 
		<img alt="Equation 1" src="/files/ftp_upload/62745/62745eq01.jpg" /></li>
	</ol>
	</li>
	<li>Delayed memory
	<ol>
		<li>Return fish, housed individually for easy identification and recording of their initial testing values, to the animal facility immediately following the initial testing period.</li>
		<li>Allow fish 4 days (or the experimental timeframe in question) between the initial testing and injury and/or delayed memory testing.</li>
		<li>Induce injury by the preferred damage paradigm (such as the modified Marmarou weight drop to induce a blunt-force trauma). House fish individually for easy identification of initial testing values, and return fish to the animal facility. 
		NOTE: Fish were injured by blunt-force TBI as previously described22.</li>
		<li>Gather 2-3 undamaged or TBI fish 4 h after initial testing and/or 4 h post-injury (or at the experimental timeframe in question) from the animal facility.</li>
		<li>Keep all fish in the dark room in individual tanks containing system water, and place one at a time in the center of the shuttle box (prepared with system water as described in 1.1), secure the lid, attach the power supply, and allow fish 15 min to acclimate.</li>
		<li>Following acclimation, assess immediate memory (strictly pass/fail) by applying only the light stimulus for up to 15 s and record the following responses:
		<ol>
			<li>Consider the trail successful if the fish crosses over the halfway point of the box within the 15 s testing period. Terminate the light stimulus upon crossing the halfway point.</li>
			<li>Consider the trail as failed if the fish does not cross over the halfway point of the box within 15 s of starting the light stimulus, terminate the light stimulus. 
			NOTE: During this post-injury testing, an adverse shock stimulus is not applied following a failed attempt.</li>
		</ol>
		</li>
		<li>Repeat step 2.4.6, with a 30 s rest period between trials, and record the number of successful trials (2.4.6.1) and failed trials (2.4.6.2) across 25 trials.</li>
		<li>Calculate the percent difference in successful trials of post-injury to the initial testing period with the equation: 
		<img alt="Equation 2" src="/files/ftp_upload/62745/62745eq01.jpg" /></li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Cognitive impairment can significantly and negatively impact the quality of life. Because of the increased visibility and occurrence of concussions and traumatic brain injuries throughout the population, it is important to understand how they cause cognitive impairment and how the damage can be minimized or reversed. For these reasons, model organisms that can be tested for cognitive decline play a critical role in these studies. Rodents have long been the primary model to investigate neurobehavior and cognition, however...

Learning and memory are routinely used as metrics of cognitive impairment, which happens due to aging, neurodegenerative disease, or injury. Traumatic brain injuries (TBIs) are the most common injury that results in cognitive deficits. TBIs are of growing concern because of their association with several neurodegenerative disorders, such as frontotemporal dementia and Parkinson&#39;s disease1,2. In addition, the increased beta-amyloid aggregations observed in some TBI patients suggest that it may also be associated with the development of Alzheimer&#39;s disease3,4. TBIs are often the result of blunt-force trauma and span a range of severities5, with mild brain injuries (miTBI) being the most common. However, miTBIs are often unreported and misdiagnosed because they result in minor cognitive impairments for only a short period, and the injured individuals usually recover fully6. In contrast, repeated miTBI events have been a growing concern because it is highly prevalent in young and middle-aged adults, can accumulate over time7, can impair cognitive development, and exacerbate neurodegenerative diseases1,2,3,4,5, similar to individuals who experience either a moderate or severe TBI8.
Zebrafish (Danio rerio) is a useful model for exploring a variety of topics in neuroscience, including the ability to regenerate lost or damaged neurons throughout the central nervous system9,10,11,12,13. Neural regeneration was also demonstrated in the telencephalon, which contains the archipallium in the dorsal-inner region. This neuroanatomical region is analogous to the hippocampus and is likely required for cognition in fish and for the short-time memory in humans14,15,16. Furthermore, zebrafish behavior has been extensively characterized and cataloged17. Learning has been studied through various techniques, including habituation to the startle response18, which can represent a rapid form of non-associative learning when performed in short blocks and with attention to the rapid decay time19. More complex tests of associative learning, such as T-boxes, plus-mazes, and visual discrimination20,21 are used but often are time-consuming, require days or weeks of preparation, and rely on shoaling or positive reinforcement. Here, we describe a rapid paradigm to assess both associative learning and either immediate or delayed memory. This shuttle box assay uses an aversive stimulus and negative reinforcement conditioning to assess cognitive deficits and recovery following blunt-force TBI. We demonstrate that undamaged control adult zebrafish (8-24 months) reproducibly learn to avoid the red light within 20 trials (&#60;20 min of assessment) in the shuttle box, with a high degree of consistency across observers. Additionally, using the shuttle box we demonstrate that learning and memory abilities across adult (8-24 months old) are consistent and are useful for assaying cognition with significant impairments between either different TBI severities or repeated TBI. Furthermore, this method could be rapidly employed as a metric to track a wide range of disease progressions or efficacy of drug interventions impacting maintenance or recovery of cognition in adult zebrafish.
Here, we provide an instructional overview of a rapid cognitive assessment that can examine both complex associative learning (section 1) and memory in terms of both immediate and delayed memory.This paradigm provides an assessment of the short and long-term memory of a learned associative cognitive task (section 2).

<table><tbody><tr><td>Flashlight</td><td>Ultrafire</td><td>9145</td><td></td></tr><tr><td>Instant Ocean</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Large DNA Gel Box</td><td>Fisher Scientific</td><td>FB-SB-1316</td><td>Shuttle Box</td></tr><tr><td>Power Supply</td><td>Fisher Scientific</td><td>FB-105</td><td></td></tr></tbody></table>

shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

The learning paradigm, outlined in the protocol and schematic (Figure 1), provides a rapid assessment of cognition with respect to associative learning. In addition, this paradigm has a high level of stringency, by defining learning as a repeated and consistent display of 5 consecutive positive trials. This paradigm is also applicable to a range of ages and injuries. Undamaged fish at 8 months (young adult), 18 months (middle-aged adult), and 24 months (elderly adult) required a similar numb...

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Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

shuttle-box-assay-as-an-associative-learning-tool-for-cognitive

Research

JoVE Journal

Neuroscience

4.4K Views.  University of Notre Dame. De Shuttle Box Assay volgt reproduceerbaar de progressie en het herstel van cognitieve stoornissen na een stomp krachttrauma of een ander type hersenletsel bij zebravissen. De Shuttle Box Assay maakt het mogelijk om relatief eenvoudig, snel en robuust associatief leren in zowel het korte- als langetermijngeheugen bij zebravissen te meten. Bereid om te beginnen de Shuttle Box voor door een gelbox van 30,5 bij 19 bij 7,5 centimeter aan te passen met een stuk plexiglas van 5 bij 19 centimeter da...

Video: Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.

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

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system ...

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

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

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 ...

Behavior

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

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed. 
This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

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

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

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely ...

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

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

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

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

Medicine

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

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

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

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Biology

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96&#160;mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p &#60; 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

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

Source: Richendrfer, H., et al. <a href="https://www.jove.com/v/50622/" target="_blank">Automated High-throughput Behavioral Analyses in Zebrafish Larvae</a>. &#160;J. Vis. Exp.&#160;(2013)
The video describes the avoidance and thigmotaxis assay of zebrafish larvae. The video examines the behavior of zebrafish larvae in response to visual aversive stimulus.

Source: Richendrfer, H., et al. Automated High-throughput Behavioral Analyses in Zebrafish Larvae. &#160;J. Vis. Exp.&#160;(2013)
The video describes the ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

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