The aim of the experiment is to assess the behavioral sensitivity of adult zebrafish to an injection of acetic acid, a noxious substance.
After injection of acetic acid, the animal is filmed for 10 min, and their behavior is recorded manually into event recorders (e.g., EthoLog). Two main variables (total locomotion and number of tail-beating events) are analyzed. To reduce variability, the behavior of the animal can be recorded before acid injection. Nociception is quantified as an increase in tail-beating and a decrease in locomotion in relation to the baseline.
The observation tank is made of either acrylic, polypropylene, or glass, with 10 cm l × 10 cm w × 20 cm h. The tank should be filled with water from the home tank, and lit from above. Contrast between fish and background can be increased by covering three walls with a white opaque material, such as white craft rubber or self-adhesive paper. A video recorder should be set up on the side of the tank, filming the non-covered wall.
Acetic acid should be diluted to 5% in Cortland's salt solution and kept on ice throughout the experiment.
- In order to reduce handling and netting stress effects, animals should be handled in a fish net daily, for at least one week, prior to starting the experiment. Refer to the Aggression protocol for more information on handling and acclimation.
- On the day of the experiment, prepare the acetic acid solution, and position the camera for recording.
- Fish are placed singly into the observation tank, and their locomotor activity recorded for 10 min.
- The animal should be quickly and gently removed from the observation tank, anesthesized with ice water or tricaine, and placed on its side on a paper towel. 5 ml acetic acid is then injected in the tail, near the caudal fin.
- The animal is returned to the observation tank. Recording should restart immediately after the animal recovers from the anesthetic plane, and continue for 10 min
- Animals which have not recovered from anesthesia in 120 s, discard its data.
- The films should be analyzed blindly (so that treatment and/or genotype is unknown to the observer) and by two independent observers. Event-recording software can be used to register tail-beating events.
- A tail-beating is defined as a movement with the caudal fin that does not lead to propulsion in the water.
Total locomotion is reduced, in relation to the baseline, by the acetic acid injection. Tail-beating events are increased by acetic acid. These endpoints are reduced (tail-beating) or increased (hypolocomotion) by analgesic treatments.