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

James Hentig; Kaylee Cloghessy; David  R. Hyde

doi:10.3791/62745

JoVE Journal > Neuroscience

Neuroscience

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

Published: July 12, 2021

doi:

10.3791/62745

James Hentig^1,2,3, Kaylee Cloghessy^1,2,3, David R. Hyde^1,2,3

¹Department of Biological Sciences,University of Notre Dame, ²Center for Zebrafish Research,University of Notre Dame, ³Center for Stem Cells and Regenerative Medicine,University of Notre Dame

Summary

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.

Abstract

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.

Introduction

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'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'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 (<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).

Protocol

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.

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° angle. Make a line marking the halfway point of the tank to assess when fish have crossed the middle of the tank (Figure 1B).
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 °C every h or after testing 3 fish.
Place 2-3 fish into a holding tank containing system water, located in a dark room where the shuttle box assay will be performed.
1. 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.
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's presence. Successful acclimation can be considered when the fish freely explores the tank.
1. 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.
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.
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.
1. 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).
2. 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.
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.

2. Memory paradigm (Figure 1A)

NOTE: This paradigm provides an assessment of the short and long-term memory of a learned associative cognitive task.

Training Period
1. 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 °C every h or after testing 3 fish.
2. 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.
3. 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.
4. 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's presence. Determine successful acclimation when the fish is freely exploring the tank.
5. 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.
6. 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.
7. 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.
8. 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).
Initial testing
1. 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.
2. Apply only the light stimulus for up to 15 s and record the responses as follows.
  1. 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.
  2. 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.
3. 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.
Immediate memory
1. 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.
  NOTE: Fish were injured by blunt-force TBI as previously described²².
2. 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.
3. 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.
4. 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.
  1. 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.
  2. 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.
5. 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.
6. Calculate the percent difference in successful trials post-injury to the initial testing period using the equation:
Delayed memory
1. Return fish, housed individually for easy identification and recording of their initial testing values, to the animal facility immediately following the initial testing period.
2. Allow fish 4 days (or the experimental timeframe in question) between the initial testing and injury and/or delayed memory testing.
3. 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.
4. 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.
5. 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.
6. Following acclimation, assess immediate memory (strictly pass/fail) by applying only the light stimulus for up to 15 s and record the following responses:
  1. 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.
  2. 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.
7. 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.
8. Calculate the percent difference in successful trials of post-injury to the initial testing period with the equation:

Representative Results

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 number of trials to learn the behavior of avoiding the red light (Undamaged 8 m: 15.28 ± 4.92 trials, 18 m: 17.66 ± 5.5 trials, 24 m: 16.2 ± 4.79 trials, 8 m vs. 18 m p=0.92, 8 m vs. 24 m p=0.98, 18 m vs. 24 m p=0.97, Figure 2A). We also utilized a severe blunt-force traumatic brain injury (sTBI) model²² and observed that fish at different ages required similar number of trials to master the assay across 1-5 days post-injury (dpi; 8 m vs 18 m, p=0.09, 8 m vs 24 m, p=0.96, 18 m vs 24 m, p=0.12, Figure 2A). At Day 1 following sTBI, fish of all ages (8, 18, and 24 m) required a similar number of trials to learn the behavior (8 m: 73.3 ± 9.45 trials, 18 m: 79.33 ± 6.35 trials, 24 m: 68.25 ± 6.65 trials, 8 m vs. 18 m p=0.71, 8 m vs. 24 m p=0.76, 18 m vs. 24 m p=0.28, Figure 2A) and they were all significantly greater than the undamaged controls (p<0.01). Collectively, these data demonstrate that the shuttle box can be utilized to examine injury-induced cognitive deficits across age ranges and suggest that adult zebrafish can recover cognitively following blunt-force injury.

Because repeated miTBI events can increasingly impair cognitive function, we used the shuttle box assay as a metric to track dose-dependent progression using repetitive TBI. We employed this assay to assess learning following a miTBI blunt force injury²² that is repeated daily for the different lengths of time. As previously observed, undamaged fish rapidly mastered the shuttle-box achieving 5 consecutive positive trials in 16.4 ± 3.5 trials (Figure 2B). One day following a single miTBI, fish display a significant increase in the number of trials to learn the behavior (40.25 ± 12.65 trials, p<0.05, Figure 2B). This deficit increased after 2 miTBI events (48 ± 14.9 trials) and was further elevated after 3 miTBI injuries (56.63 ± 12.75 trials, Figure 2B). Additionally, we observed a significant increase in cognitive impairment between miTBI fish which received a singular injury and 3 injuries (p<0.05).

We also examined how memory was affected following repeated miTBI events using the protocol for immediate and delayed memory paradigms (Figure 1A). Naïve undamaged fish were given a training period and an initial testing period, after which a portion of fish were injured for immediate memory and others were returned to the fish facility for 4 days to access delayed memory (Figure 2C). Undamaged fish exhibit a slight increase in the percent difference of successful trials in both immediate memory (6.22% ± 4.7%) and delayed memory (6.13% ± 5.57%) relative to the initial testing period. We, then examined the effect of multiple blunt-force TBI events had on memory. Significant deficits were observed following miTBI in immediate memory, but not in delayed memory. Following a single miTBI, fish displayed significant immediate memory deficits (-26.77% ± 8.93%) compared to undamaged fish (p<0.0001, Figure 2C). This trend continued with repeated injury with increasing deficits following both 2x miTBI (-37.42% ± 10.01%) and 3x miTBI (-39.71% ± 11.39%). Furthermore, we observed a similar dose-effect between fish treated with a single (1x) miTBI and 3x miTBI (p<0.05, Figure 2C). These data suggest that learning and memory is reduced in miTBI fish with the increasing number of injuries, significantly increasing the deficit and the shuttle box assay and protocols described above are sensitive enough to detect these differences.

Figure 1: The Shuttle Box Assay. (A) Instructional overview of the learning and memory paradigms for cognitive assessment. (B) Schematic of a converted large DNA gel box for the shuttle box assay. (C,D) Graphical representation of stimuli application during trials. Please click here to view a larger version of this figure.

Figure 2: Zebrafish display cognitive deficits following blunt-force TBI. (A) Following sTBI, zebrafish at 8, 18, and 24-months of age exhibit learning deficits that are not significantly different between age groups. Significant increases in the number of trials to learn the shuttle box paradigm compared to age-matched controls were observed at 1 dpi returning to undamaged levels by 4-5 dpi. (B,C) Repeated miTBI fish displayed both learning (B) and memory (C) deficits in a dose-dependent manner. The mean ± SEM is plotted in A and B, while the mean ± Standard deviation is plotted in C. Each data point on all three graphs represents a single adult zebrafish. Statistical analyses were performed with either a One-Way or Two-Way ANOVA followed by a Tukey post-hoc test. # p<0.05, ## p<0.01. Please click here to view a larger version of this figure.

Discussion

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, zebrafish have emerged as a useful model with numerous distinct behaviors to investigate a range of developmental, age-related, and acquired cognitive deficits¹⁷^,²⁰^,²³^,²⁴^,²⁵^,²⁶. Various methods to assess cognition have been utilized from one-dimensional learning in the form of habituation, to complex learning and spatial memory, novel object and location recognition, and decision making¹⁸,¹⁹^,²⁰^,²¹^,²⁷^,²⁸. However, these cognitive tests are limited to testing non-associative cognition or require a complex set-up, financial investment in equipment, or an extensive time commitment before tests can be performed. In contrast, the shuttle box and the learning and memory paradigms described here utilize a complex associative learning assay that is cost-effective, a rapidly assessed, and easily employed by a novice investigator. Most importantly, consistent with the other cognitive tests, our assay demonstrates that undamaged fish rapidly learn the associative task and can memory the task days later without intermittent training²⁹.

The adaptability of the assay provides avenues to investigate various time points of learning and memory as a metric of disease progression or mechanistic interventions. There are two primary features of the assay. First, the method is simple. The assay is quickly set up and has clear and distinct end points with respect to successful and failed trials, making it accessible to a range of investigators. We found that because of the simplicity of this assay, there is very little troubleshooting needed to use the shuttle box successfully. Second, the assay is extremely quick in comparison to other cognitive exams, which provides flexibility or the ability to examine a large number of fish rapidly in a single day. The time to assess learning is at a minimum 19.75 min (Figure 1), with the fish requiring 15 minutes to acclimate to the shuttle box (determined by tank exploration), followed by a single failed trial (15 s light stimulus, 15 s aversion stimulus, 30 s between trials) and 5 immediate and consecutive positive trials (<15 s light stimulus). In practice, we observed that undamaged fish require 6-30 trials (19.75 min-43.75 min), while in extreme cases (following a severe blunt-force trauma), the most severe deficits can require 100 trials (113.75 min). Memory studies are also rapidly performed. Following the protocol outline, the minimum time necessary for acclimation, training, and initial testing is 67.5 min (15 min acclimation, 25 iterations of light and shock for 15 s, 30 s rest between trials, and repeat for initial testing without the adverse stimuli). While retesting either immediate or delayed memory requires only 33.75 min (15 min acclimation, 25 iterations of only light stimulus for 15 s, and 30 s rest between trials), regardless of injury, treatment, or cognitive deficit.

When assessing neurobehavior, various paradigms utilize either positive or adverse stimuli. Positive stimuli in the form of food or social interaction, often used in classical T-box mazes, can aid in a strong response of a learned task. However, assays utilizing positive association do so at the expense of time. In contrast, while conditioning in response to an adverse stimulus provides a rapid association and strong behavioral response, it is at the expense of the adverse stimulus. Undamaged fish often learn the shuttle box assay quickly and are therefore subjected to a minimal number of shocks, and as a result seem to have no adverse events. However, neurologically compromised fish (TBI), with severe cognitive deficits, require a significant number of trials and electrical shocks. These multiple shocks have been observed to occasionally result in tonic-clonic seizures. Any fish experiencing a tonic-clonic seizure while within the shuttle box should be immediately removed and ethically euthanized. All trials for the euthanized fish, up to and including the seizure event, should be excluded in any statistical analysis. Furthermore, it is worth noting that electrical shock to a neurologically damaged subject could impose unintended differences between damaged fish that are and are not resulting from the shuttle box. For that reason, we suggest all fish subjected for neurobehavior assessment should not be used for any other quantitative metric (serum biomarker, IHC, etc.). It is also important to understand that this method of learning is based on a visual stimulus and is not appropriate for damage that may compromise visual circuits, as it will confound the results.

Our results demonstrate that following blunt-force TBI, zebrafish exhibit a rapid cognitive deficit that results in increased trials to master an associative task in the shuttle box assay. Similar immediate deficits are seen in rodent models of TBI, however these deficits can diminish, they often persist and remain significant³⁰. In contrast, zebrafish display cognitive recovery within 7 days following injury. The regenerative capacity of the adult zebrafish is well documented⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴^,¹⁵, with known neurogenic niches in the ventricular/subventricular zones of the telencephalon³¹^,³². The cognitive recovery observed in our assay following TBI provides insight into needed exams to identify if these neurogenic niches are stimulated and play a role in tissue and cognitive recovery.

In conclusion, the shuttle box provides a rapid assessment of cognition in regard to associative learning and memory. The assay utilizes minimal and conventual equipment and is technically simple. Future applications could be utilized to assess genetic and pharmacological interventions to neurologically insulted fish in regard to neuroprotection as well as other injury paradigms or neurodegenerative models.

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Flashlight	Ultrafire	9145
Instant Ocean	Instant Ocean	SS15-10
Large DNA Gel Box	Fisher Scientific	FB-SB-1316	Shuttle Box
Power Supply	Fisher Scientific	FB-105

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Hentig, J., Cloghessy, K., Hyde, D. R. Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish. J. Vis. Exp. (173), e62745, doi:10.3791/62745 (2021).

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