The overall goal of this procedure is to measure the visual acuity in adult zebra fish using the optokinetic response or OKR. This is accomplished by first building the OKR device that consists of a rotating drum and data capturing apparatus. Next, the fish is immobilized within the device and an OKR is elicited.
Then the binocular and monocular spatial acuity thresholds are determined using different sized gradings and a modified staircase approach. Finally, the visual acuity is calculated for the fish. Ultimately, results can be obtained that show visual acuity changes within a population of fish as they age or in diseased treated fish through the optokinetic response.
The main advantage of this technique over existing methods, such as the automotive response or OMR, is that measuring the visual acuity using the OKR is more clinically relevant and reliable. 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%trica 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 grading 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 grading, pause the rotation briefly and replace the grading with a smaller grading with a higher spatial frequency.
Repeat this process with sequentially smaller gradings until an OKR can no longer be elicited. To verify the loss of OKR following a modified staircase approach, retest with the smallest grading that caused an OKR and then with the grading that failed to elicit a response. To verify the loss of the OKR to obtain monocular acuity measurements.
Place a black plastic occlude over the stripes adjacent to the opposite eye and repeat the data collection process. Then switch to the other eye by repositioning the udder and taking measurements. Use the reference measuring strip beneath the fish tank to take note at 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 grading, and H is the length of one cycle of the smallest grading at which OKR was observed. For combined visual acuity measurements, use an average a value calculated from both the left and right eye. The procedure demonstrated in this video can determine visual acuities for zebrafish of many ages.
The best binocular spatial acuity recorded was 0.74 CPD as shown here. Binocular acuity generally increases with age, but levels off after about 12 months of age when the visual acuity was measured in five month old wild type fish compared to age matched lrp. Two bugeye fish that of asymmetric eye development.
We found that the LRP two bugeye fish had significantly reduced acuity when compared to the binocular acuity. Binocular summation is observed similarly to human vision as demonstrated in this graph. Binocular acuity was generally five to 10%better than either right or left acuities measured independently for some individual fish.
The difference was a 25%improvement. After watching this video, you should have a good understanding of how to determine the visual acuity in adult zebrafish using the OKR.
