Aggression Assay: A Method to Study Aggressive Behavior of Zebrafish in Response to a Mirror
Transcript
– Start with two rectangular tanks, an acclimation tank and an aggression tank. Now cover the outer three sides of the aggression tank with a white paper to reduce external stimuli. Mark the tank externally to divide it into four equal rectangular quadrants for position tracking. Fix a mirror in the left lower quadrant angled at 22.5 degrees from vertical. The fish's reflection acts as a stimulus for aggression.
Pour water into both the tanks. The water should be pre-warmed to 27.5 degrees. Place the focal fish into the acclimation tank. This fish is the test subject for the behavioral assay. After 10 minutes, transfer the focal fish from the acclimation tank to the aggression tank via cupping. Now let the fish acclimatize for another 30 seconds.
Record the number of bites, quick lunges toward the mirror with an open mouth, the number of lateral displays, flaring of dorsal, pectoral, anal, and caudal fins in the direction of the mirror, and the amount of time spent in the quadrant near the mirror. Aggressive fish tend to spend more time near the mirror and bite the mirror more often. In the example protocol, we will observe these aggressive behaviors in zebrafish.
– Begin by obtaining a 19 liter rectangular tank measuring 30 by 15 by 10 centimeters. Outfit the tank with external opaque partitions surrounding three sides of the aggression tank, leaving the front exposed for viewing, to reduce undesirable outside stimuli. Divide the tank with external markings into four equal rectangular quadrants. Use silicone caulk to fix a mirror within the left lower quadrant, inclined at 22.5 degrees, forming a right triangle against the far left side of the tank.
Have fresh aged water pre-warmed to 27.5 to 28.5 degrees Celsius ready to add to the aggression tank in order to ensure that the temperature of the water in the acclimation tank and aggression tank are within 2 degrees Celsius of the housing rack. Cup a focal fish from the housing rack, and transfer it into a separate acclimation tank, where it will acclimate for 10 minutes.
Confirm that the video camera is focused on the aggression assay tank and press record. Then immediately transfer the focal fish via cupping from the acclimation tank to the aggression tank. Record the aggression tank for 10 minutes and 30 seconds. Then cup the focal fish from the aggression tank back into the acclimation tank.
When watching the recording after the assay is complete, wait 30 seconds after the fish has been introduced to the tank as an additional acclimation period before scoring behaviors. Finally, score a single aggression assay at least twice until the assay is scored with high agreement between iterations.
