Pentylenetetrazole Prevention Test

Zebrafish Behavioral Protocols

PENTYLENETETRAZOLE PREVENTION TEST

Caveats and Special Instructions

Prior to attempting any zebrafish behavioral experiments, you will need to review the zebrafish housing, healthcare, and handling protocols. You will also need to review the video tracking protocol to learn how to setup the camera properly to record usuable videos. Prior to testing any fish in the pentylenetetrazole prevention test, please also review these important articles which provide the critical parameters and theoretical basis for this behavioral experiment: Mussulini et al., 2013; Wong et al., 2010; Desmond et al., 2012. For a valid response in this test, zebrafish must be housed in 2.5L tanks for 3+ days prior to testing. This behavioral assay can accomodate 4 tanks of 3 fish each, for a total of 12 fish simultaneously. To dose, test, and euthanize 12 fish, you will need a minimum of 45 minutes. Additional time is necessary for preparation, set-up and clean-up time (15-30 minutes). Plan your time accordingly. Multiple sets of 12 fish may be tested consecutively, however, the behavioral tanks need to be rinsed and scrubbed (without detergent) between each test. Each dosing beaker should only be used once before a thorough cleaning.

Preparing for the experiment

1. In the week prior to the experiment, sit down with Dr. Schmoutz and review your experimental design in your lab notebook. Make sure that you can answer the following questions:
   • This experiment is a small part of a larger project. What are the hypotheses for this experiment? What do you expect to see?
   • What are the important comparisons between treatment groups that will provide us with useful data?
   • How do you know what dose of drug to use in this experiment?
2. The day before the experiment:
   • Clean and wash as many 600 mL dosing beakers as your experimental design needs (typically 12).
   • Ask Dr. Schmoutz to prepare the stock drug solutions and PTZ powder that you will need for the experiment.
   • Ensure that the camera battery is charged. Place it on the charger if necessary.
3. On the day of the experiment, ensure that four 1.8 L fish tanks are clean and dry, to be used as the behavioral tanks.
4. Grab a clean ice bucket and create two euthanasia beakers (600 mL beaker, filled with ~300 mL of wet ice and topped to 500 mL with fish water).
5. Label Eppendorf tubes with the corresponding ZF numbers for tissue collection according to the experimental design in the lab notebook.
6. Move to Room 9-321 with camera, notebook, ice bucket, PTZ solution, stock drug solutions, dosing beakers, sheet, lights, and Eppendorf tubes.
7. Add the appropriate volumes of stock solutions to each of the dosing beakers, according to your notebook. Fill each dosing beaker to 500 ml with fish water. Arrange the dosing beakers in a way so that you’ll be able to quickly transfer fish into the first four of them.

Setting up the necessary equipment

1. Clear the metal table in the Fish Room and place the riser shelf on it.
2. Mount the lights on the wall behind the riser.
3. Place a white sheet over the metal table and riser and tape the sheet to the wall, ensuring that the behavioral tanks will be filmed on a white background.
4. Place four novel tanks on the sheet-covered table and riser, two on each level, with the spouts facing away from the center. Each tank should be pulled to the front of the table/shelf to eliminate shadows cast onto the vertical white backdrop. Place cardboard dividers between the tanks to prevent the fish from making visual contact with fish in other tanks.
5. Add the appropriate volume of stock PTZ solution to each behavioral tank. Fill each tank to 700 mL with fish water.
6. Turn on the lights behind the sheet.
7. Place the tripod ~30 cm away from metal table, attach the camera and use the bubble levels to adjust the position. Check the positioning of the camera and ensure that all four tanks are completely filling the video frame. Adjust the tripod and/or tanks to eliminate shadows, glare, or cutoffs as necessary. This is crucial to ensure optimal video quality for later analysis.
8. Place a “Do Not Disturb” sign on the door.

Performing the experiment

1. Begin by gently netting three fish from their home tank and placing them into a dosing beaker. Repeat this step three times for the three other dosing beakers for a total of 12 fish. Take note of the time that each set of fish went into the beaker. Record the birth date and home tank of each fish.
2. Allow the fish to be exposed to the dosing beakers for the specified time, based on pharmacokinetics (typically 15 minutes).
3. Start the video recording using the camera.
4. Quickly but gently pour each of the four dosing beakers (drug solution and fish) into the corresponding behavioral tank. Gently place the empty beaker next to the tank.
5. Quietly exit the room and monitor the video stream on the app. Record at least 15 minutes of undisturbed behavior.

Collecting tissues

1. After 15 minutes, stop the video recording and re-enter the room. Check for any fish that jumped out during the test.
2. Pour each behavioral tank through a fine net over the sink to collect the fish. Place the fish into the icy beaker (3 fish in one beaker).
3. Allow each fish to sit in the ice-cold water for 90-120 seconds to ensure death (a cessation of operculum and voluntary movements). Optional: Snip off the head with scissors. Dissect the tissues by blunt dissection. Place the tissues into labelled Eppendorf tubes on ice.
4. Repeat steps 2-3 for each additional behavioral tank.

Cleaning up after the experiment.

1. Wash each behavioral tank with hot tap water and scrub and rinse and allow to air dry.
2. At the end of the day: i) Remove the white sheet, lights, and shelf from the metal table. Replace any husbandry items that you removed before the experiment. ii) Remove the “Do Not Disturb” sign from the door. iii) Place the Eppendorf tubes in a box on the top shelf of the -80C freezer in room 5-347. iv) Wash the dosing beakers thoroughly.