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

Protocol

1. Novel tank test setup

1. Prepare a 250 mL beaker pre-filled with fish system water, and at least two holding tanks.
2. On the morning of the test, transfer at least 10 test adult zebrafish to be used for each experimental condition (controls and experimental adults) from fish facility into a holding tank, transfer them to the behavior room, and allow them to acclimate for at least one hour.
   NOTE: A power analysis should be performed before experimentation, yet in our hands, an n = 10 is usually sufficient to detect statistical significance. Moreover, the holding tank should contain no more than five individuals per liter of water. An acclimation of one hour is sufficient as zebrafish adults have been shown to habituate within 30 minutes of a new tank. Also, behavioral rhythms are affected by circadian processes, and thus experimental replicates done on different days should be performed within the same hours. We typically perform all experiments between the hours of 11:00 am and 6:00 pm.
3. Label the tanks such that the condition or genotype of the animals is blind to the experimenter.
   NOTE: Experiments can easily be blinded to the experimenter by labeling tanks using a letter or number system (i.e., one tank is labeled 'A', another 'B', etc.). A party not involved in the experiments labels the tanks with such a system, and masks the identities from the experimenter until after post-analysis is complete.
4. Using a net, gently place a single adult in the pre-filled beaker from step 1.1. Allow the adult fish to acclimate in the beaker for 10 minutes.
   NOTE: Record the sex of the adult, as it might be important post-analysis to look for sex-specific differences.
5. After acclimation in the beaker, introduce the fish into the novel tank (set up details below) by gently pouring out the water and adult from the beaker.
   NOTE: The steps in this section describe setting up the novel tank assay. A diagram of the end product is given in Figure 1B.
   1. Place the novel tank in the middle of the table.
   2. Position the infrared lights behind the tank and place the white acrylic sheet or diffuser screen in between the tank and LED light source.
   3. Place the diffuser so that it maximally spreads the light coming from the LEDs, and the intensity of the light is enough to illuminate the novel tank. The closer the board is to the light source, the brighter the lights will be, yet the less it will diffuse. By contrast, placing the diffuser board away from the light source will reduce light intensity, but spread the light better.
   4. Fill approximately three quarters of the novel tank with fish system water.
      NOTE: System water is generated using reverse osmosis of tap water, followed by dosing such that conductivity equals 900 ± 100 µS, that pH is neutral (7.2), and that the temperature is 27 ± 1 °C.
   5. Attach the camera to the camera stand and connect the camera to the computer. Open up the video acquisition software and adjust the camera to face the front of the tank and ensure the entire novel tank can be seen and that there are no obscured areas in the video. Adjust the tank and the infrared lights such that there is sufficient and even illumination throughout the tank when observed through the camera.
      NOTE: Before proceeding to experiments, it can often be useful to perform a trial run, in which video of a fish is captured and tracking is performed. This will ensure that the setup is sufficient for experimentation of behavior.
6. After introducing the adult into the novel tank, start the camera recording, and move away from the setup to prevent additional distress to the fish.
7. After the recording has finished, remove the individual from the novel tank and place into a new holding tank.
   NOTE: A different holding tank from the one in step 1.2 should be used to prevent repeated testing on the same individuals.

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תוצאות

Figure 1. Diagram of the novel tank setup. (A) Dimensions of the 1.8 L trapezoidal novel tank as seen from the recording side of the tank. (B) Diagram of the setup including positions of the infrared lights, camera, and barriers used to minimize human interference.

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Materials

Name	Company	Catalog Number	Comments
Camera			We use Point Grey Grasshopper3 USB camera with lens from Edmund Optics.
Infrared filter	Edmund Optics
Video Acquisition Program			Use programs such as Virtualdub or FlyCapture because the acquisition framerate can be set.
Infrared LED lights
Assay tank	Aquaneering	Part number ZT180	Size: M3 1.8 liter
Stand and clamp, or standard tripod for camera
250mL beaker
Tracking software			We use Ethovision XT 13 from Noldus Information Technology