We are interested in the capacity of visual system in health and disease mouse model. Currently, we are using optokinetic reflex, or OKR in short, which is an involuntary eye movement that serves to stabilize retina images, to understand how the visual system contributes to the adaptive processes in visual innate behaviors. OKR model studies have advanced our understanding of innate behavior and plasticity by demonstrating that prolonged visual stimulation or impaired by similar ocular reflex can increase the behavior amplitude, and by investigating the underlying molecular, synaptic, and secretory mechanisms.
We combined video-oculography and computerized virtual visual stimulation to precisely quantify the OKR behavior invokes by truncating with freely changing parameters. This procedure is relatively simple and can be standardized to accommodate large-scale studies. Our protocol accurately analyzes tuning curve shapes and prefer visual features, detects subtle differences in normal and pathological conditions, and also can monitors changes in OKR due to pharmacological treatments or visual motor learning.
The high precision and the quantitative power make it possible to compare repetitive OKR measurements of the same mice in longitudinal studies and their different pharmacological treatments or under neurocircuit preservations. It also provides opportunities to study the neurocircuits and the circuit mechanisms of OKR plasticity. After anesthetizing the mouse, place it on a heating pad to maintain its body temperature.
Apply a layer of lubricant eye ointment to both eyes and cover them with aluminum foil to protect them from light illumination, then inject carprofen subcutaneously at a dose of 20 milligrams per kilogram to reduce the pain. Wet the fur on top of the skull with 4%chlorhexidine gluconate and shave the area. Disinfect the exposed scalp twice using 70%isopropyl alcohol and chlorhexidine alcohol.
Next, inject bupivacaine subcutaneously at the incision site, then remove the scalp with scissors to expose the dorsal surface of the skull, including the posterior frontal bone, parietal bone, and interparietal bone. Apply several drops of 1%lidocaine and one to 100, 000 epinephrine on the exposed skull to reduce local pain and bleeding. then scrape the skull with a Meyhoefer curette to remove the fascia and clean it with PBS.
Gently blow compressed air onto the skull surface until the moisture is gone and the bone turns whitish, then apply a thin layer of super glue to the exposed surface of the skull, including the edge of the cut scalp, followed by a layer of acrylic resin. Now, place a stainless steel head bar along the midline on top of the skull and apply more acrylic resin, starting from the edge of the head bar until its base is completely embedded in the acrylic resin. Wait for about 15 minutes until the acrylic resin hardens.
Subcutaneously inject one milliliter of lactated Ringer's solution, then return the mouse to a cage placed on a heating pad until the animal is fully mobile. After setting up the virtual drum and video-oculography, fix the animal's head on a customized stage. Adjust the tilt of the head so that the left and right eyes are leveled and the nasal and temporal corners of the eyes are aligned horizontally.
Transfer the stage with the head-fixed animal to the center of the enclosure formed by the three monitors. Open the eye tracking software and click free run, then click the run button to turn on the camera. Adjust the position of the right eye until it appears at the center of the video.
Rotate the camera arm to the left extreme end, then manually move the animal's right eye position on the horizontal plane perpendicular to the optical axis with fine adjustment of the 2D translational stage until the X corneal reflection is at the horizontal center of the image. Now rotate the camera arm to the other end, and if the X corneal reflection runs away from the center of the image, move the right eye along the optical axis with the fine adjustment until the X corneal reflection comes to the center. Repeat several times until the X corneal reflection stays in the center when the camera arm swings left and right.
Click on the calibration button and then the run button. Turn on the Y corneal reflection LED and record its position on the video, then switch to the X corneal reflection LED and record its position. To measure the radius of pupil rotation, rotate the camera arm to the left end.
Record the positions of the pupil and X corneal reflection on the video by right clicking the mouse, then rotate the camera arm to the right end. Record the positions of the pupil and X corneal reflection on the video. Based on the recorded values, calculate the radius of pupil rotation, RP, using this formula.
To develop the relationship between pupil rotation and pupil diameter, adjust the luminance of the monitors from zero to 160 candela per square meter to control the pupil size and record the pupil diameter for each luminance value. For each luminance value, measure the radius of pupil rotation eight to 10 times and record the diameter of the pupil. Then use linear regression to analyze the relationship between the radius of pupil rotation, RP, and pupil diameter, and to derive the slope and intercept of the linear model.
After fixing the head of the mouse in the rig and calibrating the eye movements, lock the camera arm at the central position. Run the visual stimulation and eye tracking software. For phototopic OKR measurement, ensure the drum grating oscillates horizontally with the sinusoidal trajectory.
For scotopic OKR, to achieve scotopic visual stimulation, cover the screen of each monitor with a customized filter made of five layers of 1.2 neutral density film and turn the room light off. Apply one drop of pilocarpine solution to the right eye and wait 15 minutes to shrink the pupil to a proper size for eye tracking under the scotopic condition. Ensure the drop stays on the eye and is not wiped away by the mouse.
Afterward, rinse the right eye with saline to thoroughly wash away the pilocarpine solution. Finally, pull down the curtain to completely seal the enclosure, which prevents stray light from interfering with the scotopic vision. Run the visual stimulation and eye tracking software.
For scotopic OKR measurement, ensure the drum grating drifts at constant velocity from left to right, the temporal nasal direction about the left eye. The OKR gain varied with the values of spatial frequency, oscillation frequency, and the direction of movement grating. The spatial frequency tuning curve of the OKR behavior peaked at an intermediate spatial frequency of 0.16 cycles per degree.
The oscillation frequency tuning curve monotonically declined as the oscillation frequency of the drum grating increased. The horizontal OKR could also be induced by gratings moving in different directions, but the strongest horizontal OKR behavior was elicited by the temporal nasal motion. Following 45 minutes of continuous OKR stimulation, the amplitude of the OKR behavior was significantly potentiated.
