1Department of Neuroscience, Erasmus MC, Rotterdam, The Netherlands, 2Department of Neuroscience, Royal Dutch Academy of Arts & Sciences (KNAW)
de Jeu, M., De Zeeuw, C. I. Video-oculography in Mice. J. Vis. Exp. (65), e3971, doi:10.3791/3971 (2012).
Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.
The following experiments were conducted in accordance with The Duch Ethical Committee for Animal Experiments.
2. Calibrating and Measuring Eye Movements Using Video Pupil-tracking
The eye tracking system captures the movement of the pupil as a translational motion. The translational motion of the tracked pupil contains a translational component due to axial difference between the rotational center of the eye and the anatomical center of the eye (i.e. center of corneal curvature), and a rotational component due to the angular rotation of the eyeball. By subtracting the reference CR from the pupil movement/position, the undesired translational component is eliminated from the signal, resulting in a translational motion that is only due to the rotation of the eyeball. Although they are often very small, this subtraction also eliminates the translations between the head and the camera. The residual isolated translational motion is converted into the angular rotation of the eyeball by the following calibration method8,12. This calibration was performed prior to any eye movement experiment.
3. Data Analysis
4. Representative Results
Video-oculography can be used to investigate various forms of oculomotor performances (i.e. optokinetic reflex: OKR; vestibulo-ocular reflex: VOR; visually enhanced vestibulo-ocular reflex: VVOR) as well as motor learning (VOR adaptation; OKR adaptation). The OKR compensates for low-frequency disturbances using visual feedback. The OKR can be induced by rotating the well-illuminated surrounding screen (Movie 1). Rotating the surrounding screen over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6° shows how the optokinetic system is a more efficient compensatory mechanism in the low-frequency range than in the high-frequency range (Figure 5A). The VOR compensates for high-frequency head movements using signals from the vestibular organs. The VOR can be induced by rotating the animal (i.e. turntable) in the dark (Movie 2). Rotating the turntable over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6° demonstrates how the vestibulo-ocular system is more efficient in generating compensating eye movements in the high-frequency range than in the low-frequency range (Figure 5A). When the optokinetic and vestibulo-ocular system act in concert, images can be stabilized on the retina over a broad range of head movements. Rotating the turntable over a frequency range of 0.2 -1.0 Hz with an amplitude of 1.6°, while the surrounding screen is well-illuminated (Movie 3) shows how the eye generates "high gain" compensating movements over the entire frequency range (Figure 5A). All these gain and phase values are typical for mice, although gender14 and strain15,16,17 differences were reported.
The independent control over the turntable and the surrounding screen enables us to confront the mice with a mismatch between visual and vestibular information. After a long-term and uniform exposure of mismatched visual and vestibular information, the VOR of the mouse will change to compensate for the altered visual input (VOR adaptation; Movie 4). Rotating the turntable out of phase (i.e. 180°) with the surrounding screen (1 Hz, 1.6°) increases the VOR gain (Figure 5B). The maximal change in VOR gain, when using a one trial learning paradigm, is often reached after 30 minutes.
Figure 1. Schematic drawing of the mouse head-and-body restrainer. The body of the mouse is restrained using a plastic cylindrical tube with a diameter of 35 mm. The head of the mouse is immobilized by connecting the pedestal of the mouse to the iron bar with two screws. The iron bar makes an angle of 30 degree in order to position the head of the mouse in the normal pitch during ambulation. *, top view of the pedestal containing two nuts.
Figure 2. Schematic drawing of the mouse video-oculography setup.
Figure 3. Calibration of the video pupil-tracking system. A) The camera is rotated several times by +/- 10° (i.e. 20 degrees peak to peak) around the vertical axis of the turntable. The tracked pupil (P) and the reference corneal reflection (CR) recorded in the extreme positions of the camera rotation are used to calculate the radius of rotation of the pupil (Rp). B) The radius of the pupil diameter is dependent on the size of the pupil. C) Example showing the effect of pupil size on pupil position during the calibration procedure (both measured in pixels (px)). D) Relationship between Rp and pupil diameter measured in a single mouse. The thirteen different pupil diameters were accomplished by altering the intensity of the surrounding light.
Figure 4. Measuring and analyzing eye movements using video pupil-tracking. A) The angular pupil position is calculated from radius of the pupil (Rp) and the position of the Pupil (P; corrected for CR position). B) Example of compensatory eye movement induced by stimulating the vestibular and visual system (visual enhanced VOR). The turntable was rotated sinusoidally at 0.6 Hz with an amplitude of 1.6°, while the surrounding screen was well-illuminated. C) Analyses of the recording shown in B). Graph shows the averaged velocity trace of the turntable (blue) and pupil (red). These averaged traces were fitted with a sinusoidal function (black).
Figure 5. Performance and learning of the oculomotor system measured in one C57Bl6 mouse. A) Eye movements are generated by rotations of the surrounding screen (optokinetic reflex: OKR, top panels), by rotating the mouse in the dark (vestibulo-ocular reflex: VOR, middle panels) and by rotating the mouse in the light (visually-enhanced vestibulo-ocular reflex: VVOR, bottom panel) with frequencies ranging from 0.2 to 1.0 Hz at an amplitude of 1.6°. The gain of the reflex was computed as the ratio of eye velocity to stimulus velocity (left panels) and phase of the reflex was computed from the phase difference between the eye velocity and stimulus velocity (right panels). B) Motor learning was accomplished by adaptively increasing the VOR using an out of phase training paradigm. The mouse was subject to a visuovestibular training paradigm in which the rotation of the mouse was out of phase (180°) with the rotation of the surrounding screen (both rotating at 1.0 Hz, 1.6°) for forty minutes. Every 10 minutes the VOR was tested (1.0 Hz, 1.6°). In this mouse the out of phase training increased the VOR gain.
Movie 1. Animation showing the paradigm that induces OKR in mice Click here to view movie.
Movie 2. Animation showing the paradigm that induces VOR in mice. Click here to view movie.
Movie 3. Animation showing the paradigm that induces VVOR in mice. Click here to view movie.
Movie 4. Animation showing the visuovestibular out of phase training paradigm that induces VOR adaptation (increase) in mice. Click here to view movie.
In order to obtain high-quality video eye movements recordings in mice several requirements are necessary. The calibration procedure needs to be performed in the above mentioned standardized matter. For example off-center calibration, when the pupil is not positioned on the vertical midline with the reference CR during the calibration procedure, will result in an underestimation of RP and consequently an overestimation of the eye movement. Furthermore, we recommend integrating the pupil size correction method in the calibration procedure12, because trials that show a very stable pupil size are very rare. Even a small stressor during the trial can already alter the pupil diameter substantially.
When designing an eye movement experiment, the following factors need to be taken into account or controlled for because they are known to affect the eye movement response: age13,18, gender14 and strain15,16,19. Furthermore, the experimental animal should have pigmented irises since pupil detection and tracking is impossible when the contrast between pupil and iris is too low, like in the BALB/c mouse. Extremely nervous or anxious animals need to be trained, prior to the experiment, to get used to the experimental set up and the restrained condition. This animal handling procedure results in less closure or semi-closure of the eyes and prevents the generation of eye fluids during the experiment, and consequently a better pupil tracking is accomplished.
Finally, acquiring and analyzing the data requires two to three hours per animal. Therefore, eye movement recordings will likely remain a specific procedure applied to selected mice and is not suitable as a high throughput screening test.
No conflicts of interest declared.
We kindly thank the Netherlands Organisation for Health Research and Development (M.D.J, C.D.Z), The Netherlands Organisation for Scientific Research (C.D.Z), NeuroBasic (C.D.Z), Prinses Beatrix Fonds (C.D.Z), The SENSOPAC (C.D.Z), C7 (C.D.Z) and the CEREBNET (C.D.Z) program of the European Community for their financial support.
|Isofluran||Rhodia Organique Fine LTD|
|Phosphoric acid gel||Kerr||31297|
|Charisma composite||Heraeus Kulzer|
|Maxima 480 light curing unit||Henry Schein|
|AC servo-controlled motor||Harmonic drive AG|
|Halogen light (20 W)||RS components|
|Power 1401 (I/O interface)||CED limited|
|Infrared emmitters||RS components||195-451|
|Zoom lens (zoom 6000)||Navitar inc.|
|Pilocarpinenitrate (minims)||Laboratoire Chauvin|