Novel Tank Diving Assay: A Method to Study Innate Stress Responses in Zebrafish

- To begin, place an infrared light behind the novel tank and a white acrylic sheet in between the tank and the light to diffuse the light and prevent glare. Now fill 3/4 of the tank with fish system water generated by reverse osmosis of tap water. Transfer one acclimatized fish from a holding tank to a beaker containing fish system water and let it rest for another 10 minutes. Next, gently pour the water and the fish into the novel tank.

Using a still frame video image to find the top, middle, and bottom areas of the tank by calculating the height of the tank and dividing it by 3, determine the time spent in each area and the total distance traveled using tracking software. An adult fish, when placed in an unfamiliar tank, usually shows a preference for the bottom half, due to stress. As the stress level reduces, it gradually swims to the top more often. Test at least 10 fish per experiment. In an example protocol, we will see the experimental setup for a novel tank diving test.

- Begin by filling a 250 milliliter beaker and at least two holding tanks with fish system water. On the morning of the test, transfer at least 10 adult zebrafish for each experimental condition into individual holding tanks before transferring all of the fish to the behavior room for acclimation for at least one hour. Label the tanks such that the condition or genotype of the animals is blind to the experimenter, and use a net to gently place a single adult into the prefilled beaker.

Allow the adult fish to acclimate in the beaker for 10 minutes before gently pouring the water and the fish from the beaker into the experimental novel tank. As soon as the fish is in the tank, begin the camera recording and move away from the setup to prevent additional distress to the fish. When the recording has finished, remove the individual from the novel tank and place the fish into a new holding tank.