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

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 disease^1,2. 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 disease^3,4. 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 diseases^1,2,3,4,5, 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 system^{9,10,11,12,13}. 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 humans^14,15,16. 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 discrimination^20,21 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.

1. 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).
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.

1. 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).
2. 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.
3. 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:
      
4. 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:
      

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 numb...

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...

Materials

Name	Company	Catalog Number	Comments
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