implantation of a head bar on top of the mouse skull

Evaluating Optokinetic Reflex Responses in Mice: A New High-Precision Protocol

In response to visual stimuli in the environment, our eyes move to shift our gaze, stabilize retinal images, track moving targets, or align the foveae of two eyes with targets located at different distances from the observer, which are vital to proper vision1,2. Oculomotor behaviors have been widely used as attractive models of sensorimotor integration to understand the neural circuits in health and disease, at least partly because of the simplicity of the oculomotor system3. Controlled by three pairs of extraocular muscles, the eye rotates in the socket primarily around three corresponding axes: elevation and depression along the transverse axis, adduction and abduction along the vertical axis, and intorsion and extorsion along the anteroposterior axis1,2. Such a simple system allows researchers to evaluate the oculomotor behaviors of mice easily and accurately in a lab environment.
One prime oculomotor behavior is the optokinetic reflex (OKR). This involuntary eye movement is triggered by slow drifts or slips of images on the retina and serves to stabilize retinal images as an animal&#39;s head or its surroundings move2,4. The OKR, as a behavioral paradigm, is interesting to researchers for several reasons. First, it can be stimulated reliably and quantified accurately5,6. Second, the procedures of quantifying this behavior are relatively simple and standardized and can be applied to evaluate the visual functions of a large cohort of animals7. Third, this innate behavior is highly plastic5,8,9. Its amplitude can be potentiated when repetitive retinal slips occur for a long time5,8,9, or when its working partner vestibular ocular reflex (VOR), another mechanism of stabilizing retinal images triggered by vestibular input2, is impaired5. These experimental paradigms of OKR potentiation empower researchers to unveil the circuit basis underlying oculomotor learning.
Two non-invasive methods have primarily been used to evaluate the OKR in previous studies: (1) video-oculography combined with a physical drum7,10,11,12,13 or (2) arbitrary determination of head turns combined with a virtual drum6,14,15,16. Although their applications have made fruitful discoveries in understanding the molecular and circuit mechanisms of oculomotor plasticity, these two methods each have some drawbacks which limit their powers in quantitatively examining the properties of the OKR. First, physical drums, with printed patterns of black and white stripes or dots, do not allow easy and quick changes of visual patterns, which largely restricts the measurement of the dependence of the OKR on certain visual features, such as spatial frequency, direction, and contrast of moving gratings8,17. Instead, tests of the selectivity of the OKR to these visual features can benefit from computerized visual stimulation, in which visual features can be conveniently modified from trial to trial. In this way, researchers can systematically examine the OKR behavior in the multi-dimensional visual parameter space. Moreover, the second method of the OKR assay reports only the thresholds of visual parameters that trigger discernible OKRs, but not the amplitudes of eye or head movements6, 14,15,16. The lack of quantitative power thus prevents analyzing the shape of tuning curves and the preferred visual features, or detecting subtle differences between individual mice in normal and pathological conditions. To overcome the above limitations, video-oculography and computerized virtual visual stimulation had been combined to assay the OKR behavior in recent studies5,17,18,19,20. However, these previously published studies did not provide enough technical details or step-by-step instructions, and consequently it is still challenging for researchers to establish such an OKR test for their own research.
Here, we present a protocol to precisely quantify the visual feature selectivity of OKR behavior under photopic or scotopic conditions with the combination of video-oculography and computerized virtual visual stimulation. Mice are head-fixed to avoid the eye movement evoked by vestibular stimulation. A high-speed camera is used to record the ocular movements from mice viewing moving gratings with changing visual parameters. The physical size of the eyeballs of individual mice is calibrated to ensure the accuracy of deriving the angle of eye movements21. This quantitative method allows comparing OKR behavior between animals of different ages or genetic backgrounds, or monitoring its change caused by pharmacological treatments or visual-motor learning.

Author Spotlight: An Accurate and Quantitative Approach to Study Visual Feature Selectivity of the Optokinetic Reflex in Mice 

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

We're interested in the capacity of retinal system in health and disease mouse model. Currently, we are using optokinetic reflex or OKR insured, which is our involuntary eye movement that serves to stabilize retinal images to understand how the retinal system contributes to the adaptive plasticity in retinal innate behaviors. OKR model studies have advanced our understanding of innate behavior plasticity by demonstrating that prolonged retinal 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 oculargraphy 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 to include shapes and prefer visual features, detects subtle differences in normal and pathological conditions, and also can monitor 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 eyes in longitudinal studies and their different pharmacological treatments or under neurosurpreterations. It also provides opportunities to study the neurosurpres and the circuit mechanisms of OKR plasticity.

Watch this Scientific Journal Video about Implantation of a Head Bar on Top of the Mouse Skull at JoVE.com

Implantation of a Head Bar on Top of the Mouse Skull  

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.

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Research

JoVE Journal

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301 Views.  University of Toronto Mississauga. 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...

Video: Implantation of a Head Bar on Top of the Mouse Skull  

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

Calibration of Mouse Eye Movements

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 0 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 8 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.

Recording Eye Movements of the Optokinetic Reflex (OKR)

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 photopic OKR measurement, ensure the drum grating oscillates horizontally with a 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 the eye tracking software.For scotopic OKR measurement, ensure the drum grating drifts at a constant velocity from left to right, the temporonasal 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 temporonasal motion. Following 45 minutes of continuous OKR stimulation, the amplitude of the OKR behavior was significantly potentiated.