Name: shuttle box assay as an associative learning tool for cognitive assessment in learning and memory studies using adult zebrafish
Uploaded: 2021-07-12T13:00:00
Description: Watch this Scientific Journal Video about Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish at JoVE.com

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.
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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...

Watch this Scientific Journal Video about Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish at JoVE.com

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 ...

The Shuttle Box Assay reproducibly tracks the progression and recovery of cognitive impairment following a blunt force trauma or any other type of brain injury in zebrafish. The Shuttle Box Assay allows for relatively simple, rapid and robust measurement of associative learning in both short and long-term memory in zebrafish. To begin, prepare the Shuttle Box by modifying a 30.5 by 19 by 7.5 centimeter gel box with a 5 by 19 centimeter piece of aquarium-grade plexiglass added to each side at a 45 degree angle.Then make a line marking the halfway point of the tank to assess when fish have crossed the middle of the tank. After adding 800 milliliters of system water to the Shuttle Box, place two to three fish into a holding tank containing system water. Leave the tank in the dark room where the Shuttle Box Assay will be performed.Next, in the dark room, place one fish in the center of the Shuttle Box. Secure the lid and attach the electrodes to a power supply. The fish should be acclimated in the dark.Turn off all lights and acclimate the fish to the Shuttle Box for 15 minutes. Successful acclimation can be considered when the fish freely explores the tank. After successful acclimation of the fish, manually shine an 800 lumen red lens flashlight approximately two centimeters from the gel box wall on the side occupied by the fish.Do not start a trial if the fish is resting next to the platinum wire. 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. Continue to provide the light stimulus until either of the following conditions for a successful or failed trial are met.Consider the trial successful if the fish crosses over the halfway point of the tank within 15 seconds of light exposure. Once the fish crosses the halfway point, stop the light stimulus immediately. If the trial failed, use an electrophoresis power supply to apply a negative shock stimulus of 20 millivolts to one ampere, alternating two seconds on, two seconds off for 15 seconds with a maximum of four shocks or until the fish passes the halfway point of the box.Then terminate both the light and negative stimulus. Prior to the training period, ensure a successful acclimation to the Shuttle Box by leaving the fish under dark conditions for 15 minutes. Then manually shine an 800 lumen red lens flashlight approximately two centimeters from the gel box wall on the side occupied by the fish.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. While the light is shining on the fish, simultaneously apply the adverse shock stimulus of 20 millivolts to one ampere, alternating two seconds on, two seconds off for 15 seconds with a maximum of four shocks or until the fish passes the halfway point of the box. Once this is achieved, terminate the light and adverse stimulus.Repeat for 25 iterations. Upon completion, allow the fish another acclimation period of 15 minutes under dark conditions. During the initial testing period, perform a successful acclimatization in the Shuttle Box for 15 minutes, then apply only the light stimulus for up to 15 seconds and record the responses.Consider the trial successful if the fish crosses over the halfway point of the Shuttle Box within 15 seconds after starting the light stimulus and stop the light stimulus immediately when the fish crosses the halfway point. Consider the trial as failed if the fish does not cross over the halfway point of the Shuttle Box 15 seconds after starting the light stimulus and stop the light stimulus after 15 seconds. During the initial testing, do not apply an adverse stimulus following a failed attempt.Perform short-term memory testing immediately following the initial testing period. Induce dramatic brain injury and wait for a period of four hours before testing. Then acclimate the fish in the Shuttle Box for 15 minutes.Assess short-term memory by applying only the light stimulus for up to 15 seconds and record if the fish crosses over the halfway point of the box before the light is turned off, considered as passed trial, or fails to cross the halfway point within the 15 seconds considered as failed trial. Repeat the above step 25 times with a 30-second rest period between each trial and record the number of successful and failed trials. Perform long-term memory testing four days after initial testing.Induce traumatic brain injury and wait for a period of four hours before testing, then acclimate the fish in the Shuttle Box for 15 minutes. Assess long-term memory by applying only the light stimulus for up to 15 seconds and record if the fish crosses over the halfway point of the box before the light is turned off, considered as passed trial, or fails to cross the halfway point within the 15 seconds considered as failed trial. Repeat the above 25 times with a 30-second rest period between each trial and record the number of successful and failed trials.The instructional overview of the learning and memory paradigms for cognitive assessment is shown here. The undamaged fish at eight 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. After utilizing the severe blunt force traumatic brain injury model, or STBI, the fish at different ages required a similar number of trials to master the assay across one to five days post-injury.On day one following STBI, fish of all ages required a similar number of trials to learn the behavior, which was significantly greater than the undamaged controls. The undamaged fish rapidly mastered the Shuttle Box, achieving five consecutive positive trials in approximately 17 trials whereas one day following a mild brain injury, or a mild TBI, fish display a significant increase in the number of trials to learn the behavior. This deficit increased after two mild TBI and was further elevated after three mild TBI injuries.Undamaged fish exhibit a slight increase in the percent difference of successful trials in immediate memory and delayed memory relative to the initial testing period. Following a single mild TBI, fish displayed significant and immediate memory deficits compared to undamaged fish. This trend continued with repeated injury with increasing deficits following both two mild TBI and three mild TBI.The timing of applied adverse stimuli is critical. Pairing the adverse stimuli with the light and their simultaneous removal solidifies their association and is crucial for the paradigm. The assay allows for rapid assessment or complex associative learning that can be used to investigate developmental, aging, and environmental impacts on cognitive impairment.

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Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...