Description

The MazeEngineers Zebrafish choice chamber allows for experiments similar to the commonly used five-choice serial reaction time task (5-CSRTT). During testing, lights are illuminated and the gate is raised. A food delivery apparatus delivers rewards at a fixed time schedule. The proper light is illuminated in the interval or until the correct entry is chosen. The number of trials, accuracy, omissions, and latency are usually recorded. The MazeEngineers choice chamber includes an automated gate, lights on the proximal and distal ends, and control of lights and gate using Conductor Software.

The Zebrafish 5-Choice Chamber is a novel apparatus designed to evaluate impulsivity in zebrafish. The test is based on the 5-Choice Serial Reaction Time task developed by Robbins and colleagues in the early 1980’s (Robbin, 2002) which was, in turn, an analog of the human continuous performance task. The underlying objective of the apparatus is to assess the visuospatial attention and motor impulsivity in animals. The Zebrafish 5-Choice Chamber is a successor to the Zebrafish 3-Choice Chamber which is also used as an operant behavioral assay (Parker et al., 2012).

Zebrafish models are growing in popularity due to their many advantages over rodent models. Zebrafish share a similar genetic structure with humans, and their genetic tractability and the availability of forward genetic phenotype screening also make them a great option for research. Further, their maintenance and housing are much easier than other animal models. The 5-Choice Chamber allows a fully automated process of evaluation of impulsivity in the zebrafish. Thus, allowing standardization of behavioral testing procedures and maximized data reliability.

The 5-Choice Chamber consists of a large tank that houses an inner tank. The inner tank consists of 5 light stimuli and a gate to further divide the inner tank into two areas: the magazine area and the stimulus area. The fish are trained to approach the food magazine that has an illuminated light. By manipulating the light stimulus and adding other distractions or delays, the subjects are evaluated for aspects of attention and impulse control.

Other apparatus that use the Zebrafish model include the Zebrafish Y-maze, the Zebrafish T-maze, the Zebrafish Three Chamber Choice, and the Zebrafish Place Preference Test. (For more Zebrafish testing apparatus and equipment click here).

The Zebrafish 5-Choice Chamber comprises a large tank of dimensions 42 x 49 x 15 cm and a smaller inner custom-designed tank that serves as the test area. The inner tank is constructed using opaque acrylic that is divided by an automated opaque door into the food magazine area and the stimulus area. The use of opaque acrylic minimizes external visual distractions and reflections within the unit. The distance between the food delivery area and the gate is 21 cm while the distance between the stimulus area and the gate is 15 cm. The gate is supported by two acrylic columns attached to the inner tank. The floor of the apparatus is designed to allow animal waste and surplus food to pass through. Further, the floor helps create a contrasting background to assist with the video recording and monitoring.

The stimulus area has a curved design to allow visual detection and is divided into 5 slots using dividers. Within each slot is a stimulus LED light that emits bright light different in color from that of the magazine area LED. The food magazine is equipped with an LED light and has a slot for food delivery. The food delivery unit is placed on the outside of the testing tank.

The apparatus comes with automation software to control the lights and the gate.

The subjects should be housed in tanks that are maintained at a constant temperature and pH value. It is advisable that these conditions be maintained throughout the sessions to avoid unnecessary harm or stress to the fish. It is also important to keep the water aerated and the quality of water maintained at an acceptable level. Automated tracking and video software, such as the Noldus Ethovision XTand ANY-Maze can be used with a camera placed on the ceiling above the tank to allow for the tracking and recording of the behaviors and movements of the fish. Between each subject empty the tank and thoroughly rinse it before the next use.

The pretraining and training procedures for the fish in the 5-Choice Chamber are as follows:

Habituation (Week 1)

Prior to beginning, the training allows the subjects to acclimate to the testing tank for 30 minutes for at least 7 days. Ensure that the subjects are undisturbed during this period. During this period the lights should be on, and food should be delivered as per a pre-determined schedule.

Magazine Training (Week 2)

Begin magazine training by confining the subject to the magazine area by closing the divider door. Illuminate the magazine light for up to 10 seconds with 30 seconds intervals and allow the fish to consume the food by entering the magazine. Perform five 30-minute sessions to train the fish to associate the magazine light with food delivery.

Stimulus Light Training (Week 3)

Begin training by confining the subject to the magazine area. After 1 minute has passed, raise the gate to reveal the five-stimulus area with all the lights illuminated continuously. Turn off the light corresponding to the stimulus aperture when the fish enter it and illuminate the magazine light. If the subject re-enters the food magazine, lower the gate to confine it to the food delivery area and deliver food once it enters the magazine. Repeat the process 5 more times in 30-minute sessions with 20 seconds interval between each trial.

5-Choice Serial Reaction Time Training (Weeks 4 onwards)

Begin the 5-Choice Serial Reaction Time training (5-CSRTT) by confining the fish in the food delivery area for 1 minute. Raise the gate to introduce the subject to the stimulus area. After a pre-set pre-stimulus interval (PSI, the delay between the gate being raised and the stimulus being illuminated), illuminate one of the 5 stimulus lights. If the fish approaches the correct stimulus aperture (corresponding to the light) during the 30-second stimulus, illuminate the magazine light. Close the gate behind the fish once it enters the food magazine and delivers the food. Confine the subject in the food area for 20 seconds. Should the subject fail to approach the right stimulus (incorrect trial) or shows no response within the 30-second duration or approaches the aperture prior to the light being illuminated (anticipatory response), switch off the lights and confine the subject in the food area with no food for 20 seconds.

Evaluation of Impulse Control

For the experiment, 4-month-old first-generation ENU mutagenized Tübingen long-fin fish were used in the 5-CSRT task. The subjects underwent 5-CSRT training at 5 second PSI for weeks 4 to 8 and at 10 seconds PSI for weeks 9 to 11. The data obtained during the magazine training showed little evidence favoring an increase in entries into the magazine during the light exposure period. During the stimulus light training period, fishes showed a steady increase in the number of reinforcers received. For the 5-CSRTT period, a steady increase in correct responses could be observed in fishes for the 5 second PSI period. Further, increased latency of correct response and a significant speed-accuracy trade-off could also be observed during this period. On comparison of the last four days of each PSI session, a significant increase in correct responses was observed.  (Parker et al., 2013)

Evaluation of the effects of atomoxetine on 5-CSRT task performance

Adult, mixed-sex, wild-type (TU strain) zebrafish were used to analyze the effects of atomoxetine, a drug used in the treatment of ADHD, on anticipatory responses in the 5-Choice Chamber task. Subjects were administered either atomoxetine (0.5 (0.15 mg/kg), 1 (0.3 mg/kg), and 2 μM (0.6 mg/kg)) or methylphenidate (5 (1.3 mg/kg), 10 (2.6 mg/kg), and 15 μM (4 mg/kg)). The drugs were dissolved in aquarium-treated water, and the subjects were incubated in these tanks 30 minutes prior to testing. The training phase was divided into 3 phases. Subjects advanced to the next phase only if they successfully performed 20 or more trials in each session for a minimum of three consecutive days. The 5-CSRT training for weeks 4 to 5 had a PSI of 1 second while weeks 6 to 9 used a PSI of 5 seconds. Subjects that passed the first two phases proceeded to the third phase that incorporated the drug trials. A dose-dependent effect of atomoxetine on the anticipatory responses could be observed especially at2 μM atomoxetine subjects showed reduced anticipatory responses relative to baseline. On the other methylphenidate resulted in an increase of anticipatory response at 15 μM relative to the baseline. (Parker et al., 2014)

The following data can be collected during the 5-Choice Chamber task.

• Number of trials
• Number of correct responses
• Number of incorrect responses
• Latency to initiate the task
• Latency to enter the magazine
• Latency of correct responses

The general testing parameters of response rates are calculated as follows,

Strengths

The 5-Choice Chamber effectively adapts the classic 5-Choice Serial Reaction Time Training task used in rodents for use in fishes. Automation of the apparatus enables the increase of throughput and decreases the need for investigators during the task. Thus, increasing the reliability and the quality of the research. The design allows easy management of the number and duration of stimuli to manipulate the attentional load as required. Further, the use of opaque construction of the testing chamber minimizes unnecessary distractions. The apparatus shows great potential in investigations related to hyperactivity and motor impulsivity and the associated treatments.

Limitations

Further refinement of the 5-Choice Chamber apparatus may be required. The performance of the fish may be affected by disturbing external stimuli such as noise, vibration, and visual stimuli. Handling, habituation and rearing environments can also have an impact on performance. Undue stress and anxiety can result in incorrect results and observations. It is vital to maintain a constant tank temperature and other environmental factors during the test to avoid unnecessary influences on the performance. Care should also be taken to consider the health of the fish. Impulsivity and hyperactivity can be affected by several factors. The task may be time-consuming, and the consistency of the results across different labs can be affected by the type and amount of food used.

• Zebrafish 5-Choice Chamber is based on the conventional 5-Choice Serial Reaction Time Training apparatus often used in rodents.
• The task is used in the investigation of motor impulsivity and hyperactivity.
• The task is also useful in the evaluation of the effects of pharmacological manipulations on the hyperactivity and impulsivity of the subjects.
• Zebrafish serve as an ideal organism model due to their similar genetic structure to humans and their genetic tractability and the availability of forward genetic phenotype screening.
• Impulsivity and hyperactivity can be affected by several factors.
• The 5-Choice Chamber task can be time-consuming.