The overall goal of this procedure is to perform video o iconography in mice. This is accomplished by first equipping the mouse with a pedestal construction on his skull, which allows the immobilization of his head in a special head body restrainer. The second step is to place the mouse in the video ocul setup and to calibrate the video pupil tracking system.
Next eye movements are recorded while the oculomotor system is activated using a large repertoire of a vestibular and optic kinetic stimuli. The final step is to analyze these eye movements. Ultimately, video O iconography on normal, pharmacologically treated or genetically modified mice can be used to explore the physiology of motor behaviors.
Though this method can provide insight in the ocular motor system, it can also be applied to study diseases with a cerebellar vestibular or ocular origin by using mouse mutants that mimic human pathologies. To begin this procedure, anesthetize the mouse in a gas chamber with a mixture of isof, fluorine, and oxygen. Then maintain the anesthesia by delivering the gas through a mask.
Next, use a heating pad and an anal thermo sensor to maintain the mouse's body temperature at 37 degrees Celsius. Subsequently, apply the eye ointment to protect the eyes from drying, shave the dorsal cranial fur and clean the surgical area. Afterward, make a midline incision to expose the dorsal cranial surface of the skull, clean and dry the surface.
Then apply a drop of phosphoric acid from bgma to Lambda. After 15 seconds, remove the etching, then clean the cranial surface with saline and dry it again. Apply a drop of opti bond prime to the top of the etched cranial surface and air.
Dry it for 30 seconds. Next, add a drop of opti bond adhesive on top of the opti bond prime. Cure it with UV light for one minute.
After that, cover the adhesive layer with a thin layer of charisma composite. The connector with magnet screw hole, and attachment sites is embedded in the composite. Then cure the composite with UV light.
Again, if necessary, apply additional layers of composite and cure them with light. Allow the mouse to recover for at least three days after the surgery. The next step is to place the mouse in the restrainer and fix its head to the restrainer with the magnet and one screw mount the mouse head and body restrainer on an XY platform.
Using the XY platform, place the mouse's head above the center of the turntable so the mouse can be moved over the pitch Ya and roll axes. Then place its head in the correct pitch ya and roll angle by aligning the eye using the visual image of the eye generated by the eye scan system. Now the turntable is attached to an AC servo controlled motor.
The position of the turntable is monitored with a potentiometer attached to the turntable axis. The turntable is covered by a cylindrical surrounding screen with a random dot pattern, which is also equipped with an AC servo controlled motor. The position of the cylindrical screen is monitored by a potentiometer attached to its axis.
The screen can be lit by a halogen light. The movement of the turntable and its surrounding screen is controlled by a computer that is connected to an input output interface. The turntable and surrounding screen position signals are filtered by a 20 hertz cutoff frequency digitized by the input output interface and stored on this computer.
The eye of the mouse is illuminated by three infrared emitters. Two are fixed to the turntable, and the third one is attached to the camera. This third emitter produces a reference corneal reflection, which is used during the calibration procedure and during the eye movement recordings.
An infrared CCD camera equipped with a zoom lens is attached to the turntable and is focused on the mouse head. In the center of the turntable. The camera can be unlocked and moved around the turntable axis over exactly 20 degrees.
During the calibration procedure. The video signal is then processed by an eye tracking system, which can track the pupil and reference corneal reflection in horizontal and vertical direction at a sample rate of 120 hertz. Then reference corneal reference position, pupil position, and people size signals are digitized by the input output interface and are stored in the same file as the table and surrounding screen position signals to calibrate the eye movements, adjust the head position of the mouse with the camera in such a way that the video image of the pupil is situated at the middle of the monitor and the representation of the reference corneal reference is located on the vertical midline of the eye directly above the pupil.
Next, move the camera several times by 20 degrees peak. Peak around the vertical axis of the turntable. Use the positions of the tracked pupil and the reference corneal reference recorded in the extreme positions of the camera to calculate the radius of the pupil's rotation.
Repeat these steps many times under various illumination conditions in order to determine the relationship between the pupil size and pupil's rotation. Then compose a pupil's rotation correction curve. Now calculate the angular position of the eye by measuring the reference corneal reference position, pupil position, and the pupil size.
Pupil rotation value can be extracted from the pupil's rotation correction curve and the angular position of the eye can be calculated by using the following formula. The VVOR eye movement experiment is demonstrated here. Now, convert the eye positions, table positions, and surrounding screen positions into angular positions.
Next, differentiate and filter the angular positions of the eye table and surrounding screen with a Butterworth Lowpass filter. Using a cutoff frequency of 20 hertz sub, remove the secod from the eye velocity signal using a detection threshold of 40 degrees per second. Then average the table and eye velocity signals using each individual cycle in the trial fit the averaged signals with an appropriate function.
In general, a sinusoidal velocity stimulation is used and the average cycles are fitted with a sign or cosine function. This movie shows how the eye movements are generated by the rotation of the surrounding screen to cause optokinetic reflex rotating the surrounding screen over a frequency range from 0.2 to one hertz with an amplitude of 1.6 degrees. The optic kinetic system of the mouse is shown to be more efficient in the low frequency range than then the high frequency range.
Here's another movie to show how the eye movements are generated. By rotating the mouse in the dark to cause the vestibular ocular reflex rotating the turntable over a frequency range from 0.2 to one hertz with an amplitude of 1.6 degrees. The vestibular ocular system of the mouse is shown to be more efficient in the high frequency range than in the low frequency range.
Here shows another movie about how the eye movements are generated by rotating the mouse in the light to cause visually enhanced vestibular ocular reflex rotating the turntable over a frequency range from 0.2 to one hertz with an amplitude of 1.6 degrees while the surrounding screen is well illuminated, that the mouse is shown to generate efficient compensating eye movements over the entire frequency range. And this movie shows how motor learning was accomplished by adaptively increasing the vestibular ocular reflex. Using an out of phase training paradigm, rotating the turntable out of phase with the surrounding screen increases the VOR gain of this mouse.
After watching this video, you should have a good understanding of how to perform a video ocul in mice.
