We are thankful to Yingtian He for sharing data of direction tuning. This work was supported by grants from the Canadian Foundation of Innovation and Ontario Research Fund (CFI/ORF project no. 37597), NSERC (RGPIN-2019-06479), CIHR (Project Grant 437007), and Connaught New Researcher Awards.

All experimental procedures performed in this study were approved by the Biological Sciences Local Animal Care Committee, in accordance with guidelines established by the University of Toronto Animal Care Committee and the Canadian Council on Animal Care.
1. Implantation of a head bar on top of the skull
NOTE: To avoid the contribution of VOR behavior to the eye movements, the head of the mouse is immobilized during the OKR test. Therefore, a head bar...

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

<ol>
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The method of the OKR behavioral assay presented here provides several advantages. First, computer-generated visual stimulation solves the intrinsic issues of physical drums. Dealing with the issue that physical drums do not support the systematic examination of spatial frequency, direction, or contrast tuning8, the virtual drum allows these visual parameters to be changed on a trial-by-trial basis, thus facilitating a systematic and quantitative analysis of the feature selectivity of the OKR beha...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2D translational stage</td>
			<td>Thorlabs</td>
			<td>XYT1</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic resin</td>
			<td>Lang Dental</td>
			<td>B1356</td>
			<td>For fixing headplate on skull and protecting skull</td>
		</tr>
		<tr>
			<td>Bupivacaine</td>
			<td>STERIMAX</td>
			<td>ST-BX223</td>
			<td>Bupivacaine Injection BP 0.5%. Local anesthesia</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>RIMADYL</td>
			<td>8507-14-1</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Compressed air</td>
			<td>Dust-Off</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Alcon</td>
			<td>Systane</td>
			<td>For maintaining moisture of eyes</td>
		</tr>
		<tr>
			<td>Graphic card</td>
			<td>NVIDIA</td>
			<td>Geforce GTX 1650 or Quadro P620.</td>
			<td>For generating single screen among three monitors</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>For maintaining body temperature</td>
		</tr>
		<tr>
			<td>High-speed infrared (IR) camera</td>
			<td>Teledyne Dalsa</td>
			<td>G3-GM12-M0640</td>
			<td>For recording eye rotation</td>
		</tr>
		<tr>
			<td>IR LED</td>
			<td>Digikey</td>
			<td>PDI-E803-ND</td>
			<td>For CR reference and the illumination of the eye</td>
		</tr>
		<tr>
			<td>IR mirror</td>
			<td>Edmund optics</td>
			<td>64-471</td>
			<td>For reflecting image of eye</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>FRESENIUS KABI</td>
			<td>CP0406V2</td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National instruments</td>
			<td>version 2014</td>
			<td>eye tracking</td>
		</tr>
		<tr>
			<td>Lactated ringer</td>
			<td>BAXTER</td>
			<td>JB2324</td>
			<td>Water and energy supply</td>
		</tr>
		<tr>
			<td>Lidocaine and epinephrine mix</td>
			<td>Dentsply Sirona</td>
			<td>82215-1</td>
			<td>XYLOCAINE. Local anesthesia</td>
		</tr>
		<tr>
			<td>Luminance Meter</td>
			<td>Konica Minolta</td>
			<td>LS-150</td>
			<td>for calibration of monitors</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks</td>
			<td>version xxx</td>
			<td>analysis of eye movements</td>
		</tr>
		<tr>
			<td>Meyhoefer Curette</td>
			<td>World Precision Instruments</td>
			<td>501773</td>
			<td>For scraping skull and removing fascia</td>
		</tr>
		<tr>
			<td>Microscope calibration slide</td>
			<td>Amscope</td>
			<td>MR095</td>
			<td>to measure the magnification of video-oculography</td>
		</tr>
		<tr>
			<td>Monitors</td>
			<td>Acer&#160;</td>
			<td>B247W</td>
			<td>Visual stimulation</td>
		</tr>
		<tr>
			<td>Neutral density filter</td>
			<td>Lee filters</td>
			<td>299</td>
			<td>to generate scotopic visual stimulation</td>
		</tr>
		<tr>
			<td>Nigh vision goggle</td>
			<td>Alpha optics</td>
			<td>AO-3277</td>
			<td>for scotopic OKR</td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Digikey</td>
			<td>TSL254-R-LF-ND</td>
			<td>to synchronize visual stimulation and video-oculography</td>
		</tr>
		<tr>
			<td>Pilocarpine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P6503</td>
			<td></td>
		</tr>
		<tr>
			<td>Post</td>
			<td>Thorlabs</td>
			<td>TR1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Post holder</td>
			<td>Thorlabs</td>
			<td>PH1</td>
			<td></td>
		</tr>
		<tr>
			<td>PsychoPy</td>
			<td>open source software</td>
			<td>version xxx</td>
			<td>visual stimulation toolkit</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>RWD</td>
			<td>S12003-09</td>
			<td>For skin removal</td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Krazy Glue</td>
			<td></td>
			<td>Type: All purpose. For adhering headplate on the skull</td>
		</tr>
	</tbody>
</table>

quantification of visual feature selectivity of the optokinetic reflex in mice

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.

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.

With the procedure detailed above, we evaluated the dependence of the OKR on several visual features. The example traces shown here were derived using the analysis codes provided in Supplementary Coding File 1, and the example traces raw file can be found in Supplementary Coding File 2. When the drum grating drifted in a sinusoidal trajectory (0.4 Hz), the animal&#39;s eye automatically followed the movement of the grating in a similar oscillatory manner (Figure 3B</...

Watch this Scientific Journal Video about An Accurate and Quantitative Approach to Study Visual Feature Selectivity of the Optokinetic Reflex in Mice at JoVE.com

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 ...

quantification-visual-feature-selectivity-optokinetic-reflex