Escape Response Assay: A Method to Study the Response of Zebrafish Larva to Touch Stimuli

1. Touch-evoked Response Assay

1. Preparation of 2 dpf Embryos for Touch-evoked Response Assay
   1. Ensure that the time of day at which the test is conducted is consistent between experiments because activity can vary dramatically throughout the day.
      ​NOTE: The experiment should be performed blinded and the order of testing randomized to minimize experimental artifacts.
   2. Assign the fish strains a number, which is unknown to the individual carrying out the experiment. Following this, using freely available online tools generate a random list that dictates the order of testing.
   3. At least one hour prior to testing, dechorionate embryos by ripping a hole in the chorion and gently pulling the chorion apart using a pair of fine tweezers. Remove any debris from the petri dish before returning to the 28 °C incubator.
2. Performing Touch-evoked Response Assay
   1. Heat stage to 28 °C at least 15 min prior to starting the testing.
      ​NOTE: This stage is temperature controlled and will remain at 28 °C for the duration of the testing. Temperature will affect activity and it is therefore important to maintain a constant temperature. If a heated stage is not available then the temperature of the water should be monitored and all experiments should be conducted at the same temperature.
   2. Place a petri dish filled with embryo medium (5 mM NaCl, 0.17mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄ in water) onto an illuminated stage and mount the high-speed camera over the petri dish.
   3. Launch the video camera recording software (such as Stream Pix 5, described here) and under the "workspace" tab select 1,000 frame per sec (1,000 fps) as the capture speed to ensure that fast swimming action of the fish is recorded.
   4. Working with one embryo at a time, place the embryo in the middle of the petri dish with the zebrafish clearly visible in the field of view.
      ​NOTE: If the embryo swims away prior to commencement of the experiment replace it with another, as recapturing and positioning of the embryo may result in it becoming desensitized to the stimulus and repeated burst responses may promote muscle weakness in some disease models.
   5. Begin recording by clicking on the "record" button and, deliver the mechanosensory stimulus to the embryo by touching it gently with a blunt needle on the top of the head.
   6. Stop the recording after the embryo has swum out of the field of view or returned to rest.
      ​NOTE: The acceleration peaks within the first 0.2 sec of the burst escape response following the mechanical stimulus. Therefore, ensure that at least during the first 0.2 sec of the escape response recording the fish is in the field of view. Using the software Stream Pix 5, the data will automatically be saved as a .avi video file. Alternative video capture software such as Free Video Capture or Softonic, both of which are freely available for download, could also be used.
   7. Return the embryo to a new petri dish and select another embryo for testing. Perform testing on a minimum of 15 fish.