This manuscript describes a simple method to measure stress behaviorally in adult zebrafish. The approach takes advantage of the innate tendency that zebrafish prefer the bottom half of a tank when in a stressful state. We also describe methods for coupling the assay with pharmacology.
The novel tank assay will combine with powerful genetic tools in Zebrafish offers a method for dissecting how the brain modulates stress and how neural circuits may be disregulated. Genetic and neuronal signaling pathways are highly conserved between Zebrafish and mammals and this technique can help further our understanding of the mechanisms underlying the stress response. The method is not specific to Zebrafish and has been used with the Mexican blind cave fish and therefore can potentially be extended to other fish species.
A trial run may be helpful for determining proper illumination and contrast of the animal against a background to achieve successful tracking. The assay's rarely used, but can be an involved process. Visualizing the steps will help other researchers successfully execute the procedure.
Jacqueline Chin, a post-doc in the lab, and Lydia Albert, an undergraduate student, will be demonstrating this procedure. 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 pre-filled 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. To assess the effects of specific drugs of interest, dilute the drug to a working concentration in 250 milliliter beakers of system water. Using a beaker with system water only as a control.
Mask the identities of the drug and control beakers to ensure that the tester is blind to the treatment conditions until post-analysis and perform a novel tank test as demonstrated to obtain a baseline behavioral stress response. At the end of the baseline recording, use a net to immediately transfer the fish into one of the test beakers for 10 minutes before transferring the fish to a wash out beaker containing fresh system water only. It is important to use separate nets designated for each treatment beaker to prevent accidental dosing of the control fish, especially when several drug treatments are involved.
After 10 minutes, place the fish into a new novel tank for a second novel tank test. At the end of the test transfer the adult Zebrafish into a new holding tank and replace the system water in the second novel tank with fresh system water for the next step to prevent cross-contamination of the experimental drug. At the end of all of the trials, return the individuals to the fish facility.
Here the locomotor activity of a single Zebrafish during the first and last minute of a 10 minute novel tank test is shown. When first introduced into the tank, the fish typically spend the majority of the time in the bottom of the novel tank. Over time however, the adult fish demonstrate a gradual increase in the amount of time spent in the top of the tank.
Indeed, the total time spent in the top of the tank during the first minute is significantly shorter compared to the last minute of the novel tank test. By contrast, the total distance traveled between the first and last minutes reveals no significant differences. In this representative experiment, analysis of the adult Zebrafish locomotor paths revealed little difference between the Zebrafish swimming activity before and after treatment for groups of adults exposed to vehicle alone.
By contrast, adults exposed to an anxiolytic drug spent a large amount of time in the top of the tank compared to the locomotor paths of the same fish before the drug exposure. These differences were not due to less locomotion in general as no significant differences in traveled distance were measured for any of the groups. As behavioral rhythms are affected by circadian processes, be sure to carry out experimental replicates performed on different days during the same time of day for each experiment.
Additional behavioral assays, such as the open field test or other physiological measurements of stress, can be performed to further validate results obtained from the novel tank diving test. This method can be used a high throughput assay for addressing questions related to stress, fear, and anxiety.
