The Zebrafish Shuttlebox introduces a novel approach for conducting high-throughput experiments in learning paradigms. In this methodology, a pair of screens are employed within an automated computer interface to generate computer-animated images of zebrafish or any image designated by the researcher. The straightforward binary choice paradigm renders it a valuable tool for conducting high-throughput assays in zebrafish research.
38 L experimental tank (50 Ć 25 Ć 30cm - width Ć depth Ć height)
2.5 cm thin; grey; corrugated; plastic frame mounted to midline; side; and bottom of glass to minimize contact with video side of glass
Price includes two monitors
Introduction
The Zebrafish Shuttle Box assessment serves as a tool for gauging the cognitive prowess of zebrafish. Employing a tank setup equipped with dual digital screens positioned at opposite ends, it delves into associative learning, temporal learning, and social behaviors exhibited by subjects in response to visual cues.
Zebrafish have emerged as prominent animal models owing to their genetic resemblance to humans and manageable upkeep requirements. Moreover, their strong social inclinations, favoring shoaling, contribute to the rationale behind the Zebrafish Shuttle Box's design. Leveraging the fish's social tendencies, subjects are inclined to approach and linger around animated depictions of conspecific groups, a phenomenon bolstered by the reinforcing properties of such visual stimuli (Al-Imari & Gerlai, 2008). By utilizing digital screens to present visual cues, researchers attain heightened control over spatial and temporal stimulus parameters, obviating the necessity for live conspecifics as visual aids.
Among other apparatuses tailored for zebrafish research are the Zebrafish Y-maze, the Zebrafish Three-Chamber Choice, and the Zebrafish Place Preference Test.
Apparatus and Equipment
The Shuttle Box comprises a glass enclosure with a volumetric capacity of 38 liters. Its dimensions measure 50 cm in width, 25 cm in depth, and 30 cm in height. To facilitate experimental procedures, a narrow corrugated plastic frame, 2.5 cm in width, is affixed to the interior sides and bottom glass at the center of the tank. Positioned on the outer surface of the tank, monitors are symmetrically situated on opposing sides.
Training Protocol
Ensure zebrafish are housed in tanks where temperature and pH remain stable. Consistently maintain environmental parameters to minimize stress and potential harm to the subjects. Oxygenate the water to uphold its quality at an optimal level. Prior to conducting tests, thoroughly clean the tank. Employ tracking systems like Noldus Ethovision XT to monitor and document fish behaviors. Notably, the Zebrafish Shuttle Box task does not necessitate habituation or pretraining.
Transition the subject from its housing tank to the Shuttle Box. Initially, present a black screen on both tank screens for a duration of 10 minutes. Subsequently, introduce the animated stimulus on one side of the tank, chosen randomly, for the ensuing 10 minutes. Conclude the session with a 2-minute display of a black screen. Randomly alternate the side of stimulus presentation for each experimental subject.
Move the subject from its holding tank to the Shuttle Box. Present the animated stimulus on one of the screens for a duration of 20 seconds (Stimulus Presentation Interval). This is followed by a 90-second Inter-Stimulus Interval (ISI) wherein no stimulus is displayed. Repeat this cycle of stimulus presentation and ISI at least 30 times. Depending on the requirements of the investigation, the stimulus can be displayed on alternating sides, the same side, or randomly.
Pather and Gerlai (2009) conducted an assessment on zebrafish of the AB strain, comparing their performance in the animated stimulus presentation task and associative learning tasks within the Shuttle Box. The stimuli comprised images of 6 female zebrafish exhibiting random movements at varying speeds. Their findings indicated similarities in response patterns between the animated stimulus presentation task and associative learning tasks. Zebrafish tended to approach the screen during stimulus presentation intervals and retreat upon stimulus removal. Notably, there was an uptick in swimming activity during the inter-stimulus interval (ISI), potentially driven by the fishās exploratory tendencies upon stimulus cessation. Additionally, they observed a notable increase in thrashing behavior near the wall adjacent to where the stimuli were displayed, indicative of a shoaling response, which decreased significantly during ISI periods.
Transition the subject from its housing tank to the Shuttle Box. Initially, present a black screen on both tank screens for a duration of 10 minutes. Subsequently, introduce the animated stimulus on one side of the tank, chosen randomly, for the ensuing 10 minutes. Conclude the session with a 2-minute display of a black screen. Randomly alternate the side of stimulus presentation for each experimental subject.
Move the subject from its holding tank to the Shuttle Box. Present the animated stimulus on one of the screens for a duration of 20 seconds (Stimulus Presentation Interval). This is followed by a 90-second Inter-Stimulus Interval (ISI) wherein no stimulus is displayed. Repeat this cycle of stimulus presentation and ISI at least 30 times. Depending on the requirements of the investigation, the stimulus can be displayed on alternating sides, the same side, or randomly.
Pather and Gerlai (2009) conducted an assessment on zebrafish of the AB strain, comparing their performance in the animated stimulus presentation task and associative learning tasks within the Shuttle Box. The stimuli comprised images of 6 female zebrafish exhibiting random movements at varying speeds. Their findings indicated similarities in response patterns between the animated stimulus presentation task and associative learning tasks. Zebrafish tended to approach the screen during stimulus presentation intervals and retreat upon stimulus removal. Notably, there was an uptick in swimming activity during the inter-stimulus interval (ISI), potentially driven by the fish's exploratory tendencies upon stimulus cessation. Additionally, they observed a notable increase in thrashing behavior near the wall adjacent to where the stimuli were displayed, indicative of a shoaling response, which decreased significantly during ISI periods.
Sison and Gerlai (2011) examined the impact of MK-801 on zebrafish learning capabilities using the Zebrafish Plus Maze. The study involved seven groups: six experimental groups and one control group. The experimental groups were exposed to MK-801 at a concentration of 20 ĀµM at three different times: before the initial training session, after the final training session, and immediately prior to the probe trial. The control group, which received 0 ĀµM MK-801, was exposed separately to the red stimulus tank (conditioned stimulus, CS) and zebrafish conspecifics (unconditioned stimulus, US). The findings revealed that MK-801 impaired memory performance in subjects after training. No memory disruption was observed when the drug was administered before training. Control fish exposed to the CS-US pairing spent 25-30% of their time in the target compartment, while those not exposed to the pairing spent only 8% of their time there. Administration of 20 ĀµM MK-801 significantly impaired memory, as indicated by the reduced percentage of time spent in the target compartment following training trials.
Data Analysis
The following parameters can be observed using the Zebrafish Shuttle Box.
Percentage of time spent on the side where the stimulus was presented
Percentage of time swimming
Motor movement pattern
Posture movement pattern
Differential fear
Distance of the fish from the screen where the image appears
Strengths & Limitations
The Zebrafish Shuttle Box task serves as a valuable tool for evaluating various cognitive processes, including learning, memory, and developmental changes in zebrafish. Its computerized nature and simplicity facilitate high throughput screening. By digitally presenting stimuli, researchers gain full control over spatial and temporal stimulus delivery, thereby enhancing precision in experimentation. This versatility allows for the assessment of diverse aspects of learning, such as fear-based learning using aversive visual stimuli. Moreover, the Shuttle Box enables the investigation of the effects of brain lesions, pharmacological interventions, and various diseases and disorders on zebrafish behavior and cognition.
In addition to video tracking, manual observations might be necessary to distinguish between various movement patterns. External stimuli could potentially induce freezing behavior in the fish during experiments, resulting in skewed outcomes. The length of the Shuttle Box might influence the interpretation of whether zebrafish are changing sides in anticipation of the stimulus or simply seeking proximity to shoal mates. Moreover, the choice of visual stimuli could impact the subjectsā task performance. Images depicting atypical or abnormal conspecifics might either be disregarded or actively avoided by the fish.
The Zebrafish Shuttle Box task serves as a valuable tool for evaluating various cognitive processes, including learning, memory, and developmental changes in zebrafish. Its computerized nature and simplicity facilitate high throughput screening. By digitally presenting stimuli, researchers gain full control over spatial and temporal stimulus delivery, thereby enhancing precision in experimentation. This versatility allows for the assessment of diverse aspects of learning, such as fear-based learning using aversive visual stimuli. Moreover, the Shuttle Box enables the investigation of the effects of brain lesions, pharmacological interventions, and various diseases and disorders on zebrafish behavior and cognition.
In addition to video tracking, manual observations might be necessary to distinguish between various movement patterns. External stimuli could potentially induce freezing behavior in the fish during experiments, resulting in skewed outcomes. The length of the Shuttle Box might influence the interpretation of whether zebrafish are changing sides in anticipation of the stimulus or simply seeking proximity to shoal mates. Moreover, the choice of visual stimuli could impact the subjects' task performance. Images depicting atypical or abnormal conspecifics might either be disregarded or actively avoided by the fish.
Summary & Key Points
The Zebrafish Shuttle Box task is used in the evaluation of learning, memory and development alterations in Zebrafish.
The Zebrafish Shuttle Box task is automated and provides researchers spatial and temporal control over the stimulus delivery.
Manual observations may be needed to discern between different types of movement of zebrafish.
Responses of the zebrafish are influenced by the visual stimuli. Presentation of images of conspecifics with atypical or abnormal features may not elicit the expected shoaling behaviors.
The length of the test tank must be considered when deriving conclusions from the data.
Extraneous cues can distract the subjects and hamper the results.
