Optokinetic Response Assay: A Method to Measure Visual Acuity in Zebrafish

- Secure an anesthetized fish on a platform with its eyes and gills extending beyond the platform's edge. Position the fish upright in a water-filled cylinder within a well-lit drum that can rotate freely. Once the fish revives, indicated by gill and eye movement, place a grating or pattern of alternating white and black vertical stripes into the drum. Now place a solid black occluder over the stripes in the view of the left eye.

Begin the rotation and record the optokinetic response, OKR, for the right eye. The OKR consists of reflexive eye movements in response to a visual stimulus. Replace the pattern with smaller and smaller black and white stripes until eye movement is no longer observed. Perform the same procedure for the left eye.

To calculate monocular visual acuity, the ability to distinguish fine details, measure the distance from the center of the lens of the eye to the pattern and the length of the smallest pattern of a pair of white and black stripe at which eye movement was observed. In the example protocol, we will use the optokinetic response to measure monocular and binocular visual acuity in adult zebrafish.

- Optokinetic studies are carried out with a custom-built recording device using a 14.5 centimeter diameter rotating drum, a stereo microscope with adjustable light intensity settings, a computer, and a camera for capturing images and recording. A computer operated microcontroller is used to adjust the rotating drum speed and spinning directions. Using fish that are maintained under standard conditions, begin an analysis by anesthetizing a fish in 0.016% tricaine for two to three minutes and then placing it on a small platform with the eyes and gills suspended over the edge.

To keep the fish immobilized while avoiding injury, place a thin sponge or towel over the body of the fish and pin two to three pieces of foam over the fish, ensuring that tail movement is limited. Position the fish within a cylindrical water-filled tank that fits inside the rotating drum of the OKR recording device. Use magnets on the platform to position the fish upright with the eyes approximately 7.3 centimeters from the edge of the drum.

Once the fish revives from anesthesia, and normal breathing and random eye movements are observed, place a base grating of 0.07 cycles per degree, or CPD, into the rotating drum and engage the computer controls to begin rotation and video capture. After an initial OKR is elicited by the base grating, pause the rotation briefly and replace the grating with a smaller grating with a higher spatial frequency. Repeat this process with sequentially smaller gratings until an OKR can no longer be elicited. To verify the loss of OKR, following a modified staircase approach, retest with the smallest grating that caused an OKR, and then with the grating that failed to elicit a response to verify the loss of the OKR.

To obtain monocular acuity measurements, place a black plastic occluder over the stripes adjacent to the opposite eye and repeat the data collection process. Then switch to the other eye by repositioning the occluder and taking measurements. Use the reference measuring strip beneath the fish tank to take note of the distance of each eye from the strip for making accurate acuity measurements.

To determine visual acuity, calculate CPD using the following formula, where a is the distance from the center of the lens to the grating, and h is the length of one cycle of the smallest grating at which OKR was observed. For combined visual acuity measurements, use an average a value calculated from both the left and right eye.