This test uses vertical distribution in a novel environment as a validated behavioural test for assessing 'anxiety-like' behaviours in adult zebrafish.
Conceptually similar to the rodent open-field and elevated plus maze tasks, the novel tank diving test utilises zebrafish’s instinctive behaviour to seek protection in novel environments. When first introduced to a novel tank, zebrafish will dive to the bottom and gradually increase their vertical swimming over time. This gradual increase in exploration is interpreted as a reduction in anxiety (Wong, 2010).
The novel tank diving test is quick to implement (5-10 minutes) and sensitive to various pharmacological, genetic, and environmental manipulations.
Adult zebrafish during a novel tank diving test. Courtesy of Dr Matt Parker's lab, University of Portsmouth
Experimental set up
The AD unit comes supplied with a novel tank diving kit, which includes a tank, a funnel, a tank stand, two stand locator strips, and a foam door.
Novel tank diving insert kit includes a tank, a stand, a fish funnel and a foam door.
To conduct the novel tank diving assay in the Zantiks AD unit, place the entire system on its side, with the covered hole facing up. Insert the funnel into the unit’s side hole, turning it slightly to help ease it in. The tank stand should then be placed into the centre of the chamber opening with the locator strips on either side to secure it in place. Make sure that the ridge on the stand’s raised platform is situated at the back of the unit, so that the tank can slide in easily. After filling the tank with water, slide it onto the stand, ensuring that it is secure on the raised platform. You can now insert the foam door to isolate the testing chamber.
With the AD unit placed on its side, the tracking camera will now have a side view of the fish’s movements in the testing tank. The ‘floor screen’ is now located behind the novel testing tank and will be used for illumination from the back of the tank, while the LED ‘ceiling lights’ are now located in front the tank and can be used to illuminate the tank from the front.
Seting up the Zantiks AD unit for a novel tank diving assay with zebrafish
Fish should be singly housed for one week prior to behavioural testing. If not already housed in the experimental room, allow one hour to for the fish to acclimatise the experimental room. The novel tank should be filled with water up to 13cm deep. Fish are placed individually in the novel tank.
Run the experiment via the console before the fish is placed in the tank. Recording will begin immediately after the fish enters the experimental arena. Introduce the fish into the tank by netting it through the funnel at the top. Each trial will last 5-10 minutes. All fish can only experienced the novel tank once.
Results / data ouput
For analysis, the tank is virtually divided into 2 (top, bottom) or 3 (top, middle, bottom) equally target zones. The relative position in the zones is taken as the index of anxiety. Time spent and distance travelled in each zone, as well as entries into the zones can all be recorded automatically by the Zantiks AD unit.
Common variables measured in a novel tank diving assay include:
- Latency to enter the top (s): amount of time it takes the fish to enter into the top zone of the tank - main measure of anxiety level. The longer the fish takes to enter the top zone, the higher the anxiety level is interpreted to be.
- Time spent in zones (s): total time spent in each zone - a longer duration in the top of the tank indicates lower anxiety levels.
- Number of entries to the top: number of entries into the top zone - more top entries indicates lower anxiety levels.
- Distance travelled in the top (cm): total distance travelled in the top zone - more distance travelled in the bottom of the tank indicates higher anxiety levels.
- Total distance travelled (cm): total distance the fish has travelled throughout the entire novel tank - can reflect general motor/neurological phenotypes.
- Average entry duration (s): average time spent at the top of the tank following an entry. Calculated as time spent in the top / number of entries into the top. Shorter average entry duration indicates higher anxiety level.