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

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.