Name: Non-aversive Animal Restraint Enabling Recording of Optomotor Reflex in Ground Squirrels (Video) | JoVE Journal
Uploaded: 2025-07-25T13:40:00.000+00:00
Duration: 7 min 28 s
Description: Learn to measure optomotor responses in 13-lined ground squirrels using a non-aversive platform design. This method reduces anxiety and enables reliable visual function assessment without extensive acclimatization.

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5 minutes

This research was supported [in part] by the Intramural Research Program of the NIH, National Eye Institute. This work was also supported by the Office of the Assistant Secretary of Defense for Health Affairs and the Defense Health Agency J9, Research and Development Directorate, through the Vision Research Program under Award No. (CDMRPL-18-0-VR180205). Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. The authors thank Dr. Wenxin Ma for providing the rd10 and CX3CR1GFP/+ mice, Dr. Haohua Qian (Visual Function Core, NEI, NIH) for insightful discussion and technical assistance with the OMR, the NIMH/NINDS/NICHD machine shop for help with component fabrication, and Charles King (NIH Library) for assisting in 3D printing.

The procedures involving animals were conducted in adherence to the laws of the United States and the regulations of the Department of Agriculture. All experiments with animals were conducted according to protocols approved by the Animal and Care and Use Committee of the National Eye Institute at the National Institutes of Health. Male and female 13-lined ground squirrels (ages 6-8&#160;months) used in this study were purchased from the University of Wisconsin Oshkosh Squirrel Colony. Mice (rd10: 1 month, CX3CR1GFP/+: 6.5 months) used in this study were bred in our animal facility. Animals and their cages were randomly selected and assigned to experimental groups.
1. Preparation of the prototype platform and modified handling tunnel
NOTE: Round flat platforms are commonly employed in systems designed to measure the optomotor response13. However, hyperactive rodent models, such as squirrels, often struggle to maintain stability on these platforms. This design provides a semi-enclosed space, which helps the animals habituate quickly and reduces anxiety-like behaviors. This improvement enhances the animal&#39;s ability to focus and minimizes instances of imbalance or falls during recordings.
<ol>
	<li>In CAD drawing software (e.g., OpenSCAD or FreeCAD), design the platform (gray) and the enclosure (red) as shown in Figure 1.
	<ol>
		<li>Ensure that the key dimensions for the squirrel platform are maintained as the length and width (L x W): 129 mm x 81 mm, the thickness: 7 mm, and the height of the wall that extends around the perimeter: 8 mm. Use the provided free CAD standard document file for the prototype platform (squirrel) used in this proof-of-concept study (Supplementary Coding File 1).</li>
		<li>Ensure that the key dimensions for the removable top enclosure (looking top down) are maintained as the length (119.5 cm) and width (76.2 cm), the height of the sides (50.8 mm), and the part&#39;s thickness (3 mm). Use the provided free CAD standard document file to replicate&#160;the removable top enclosure (squirrel) employed in this proof-of-concept study (Supplementary Coding File 2).</li>
	</ol>
	</li>
	<li>Adjust the diameter of the through hole in the base of the platform as needed to fit the pedestal used to elevate the platform. The diameter pictured is 40 mm.</li>
	<li>Print the platform using a white opaque filament. This provides maximal contrast for the camera, enabling the tracking software to correctly detect the animal&#39;s contour on the platform.</li>
	<li>Print the removable top enclosure with opaque dark filament (e.g., red). This provides a sense of shelter for the rodents, which facilitates guiding them into the tunnel before they are lifted and transferred to the arena.</li>
	<li>Have a machinist cut the acrylic extruded clear square tube using a table saw so that the long end is ~ 101.6 mm in length. Be sure to use protective eye wear and a push stick to guide the acrylic through the blade while keeping your hands safe from potential injury.</li>
	<li>Using the table saw or a handheld rotary tool equipped with an abrasive cutting wheel, remove one of the long rectangular faces. This creates an open top through which the camera mounted above will have an unobstructed view of the animal&#39;s position and head movements.</li>
	<li>Clean all edges with a hand file or sandpaper. Assemble the modified handling tunnel using the Acrylic Extruded Clear Square Tube cut to length with the 3D printed removable top enclosure by placing the enclosure on top of the open face.</li>
</ol>
2. Setting up the OMR arena
<ol>
	<li>Disinfect the platform and the square tube enclosure assembly with a cleaner/disinfectant before and in between animals to remove unwanted olfactory cues. 
 NOTE: While leather gloves are used to handle the animals and come into direct contact with them, they are not worn during the recordings and do not cause any lasting or observable confounding effects.</li>
	<li>Tear a small piece of paper towel measuring approximately 50 mm x 50 mm and fold it to cover the top surface of the pedestal. Place the 3D-printed platform onto the pedestal (14 inch high) with the piece of paper towel in place. 
	NOTE: The paper adjusts the press fit of the platform, prevents unintended movement, provides a uniform white background for imaging (if the pedestal&#39;s surface is not white), and acts as an absorbent layer in case the animal urinates during the recording, preventing the platform from becoming slippery.</li>
	<li>Wearing leather work gloves, gently guide the animal into the square tube enclosure assembly.</li>
	<li>Transfer the animal to the arena by placing the enclosure assembly onto the platform (Figure 1J).</li>
	<li>Carefully detach the removable top enclosure leaving the animal in the acrylic extruded clear square tube on the platform. Close the door to the OMR system</li>
</ol>
3. Setting up the OMR software parameters
NOTE: The pictured OMR system uses a compact industrial camera featuring an IR-sensitive 1/3&#8221; CMOS sensor with a global shutter at 25 frames per second (fps). The camera is equipped with an F1.6 wide-angle lens, enabling a complete top-down view of the arena. Preliminary visual acuity tests conducted on squirrels using the standard 100% contrast setting revealed that the squirrels&#8217; visual acuity exceeded the resolution limits of the hardware. The 23.8&#8221; full HD In-Plane Switching screens used in the setup have a resolution of only 1920 pixels in width, and the software is therefore hard-coded with a maximum spatial frequency of 2 cyc/&#176;. For comparison, the visual acuity of C57Bl/6 mice is approximately 0.3-0.5 cyc/&#176;. At 2 cyc/&#176;, each stripe averages 5.3 pixels in width. With a rotation speed of 12&#176;/s and a frame rate of 60 frames per second, the pattern shifts by 0.2&#176; per frame, equivalent to 4.2 pixels per frame, assuming uniform pixel speed. At spatial frequencies near 2 cyc/&#176;, the stripe pattern approaches the resolution limit where aliasing may occur, causing the pattern to appear as if it is moving backward. To prevent this artifact, the software is hard-coded to cap the spatial frequency at 2 cyc/&#176;.
Thus, for squirrels we devised a protocol where we perform the acuity test but at a much lower contrast. We selected a contrast setting (12.78%) where the squirrels were unable to see the striped pattern at 2 cycles/&#176;.
<ol>
	<li>&#160;In the live video panel, ensure that the animal is adapting well to the new environment and is beginning to move less and calming down.
	<ol>
		<li>At 2 cyc/&#176;, each stripe averages 5.3 pixels in width. Set the rotation speed to 12&#176;/s and a frame rate of 60 frames per second; the pattern shifts by 0.2&#176; per frame, equivalent to 4.2 pixels per frame, assuming uniform pixel speed. Set the spatial frequencies near 2 cyc/&#176; so the stripe pattern approaches the resolution limit where aliasing may occur, causing the pattern to appear as if it is moving backward. To prevent this artifact, hard code the software to cap the spatial frequency at 2 cyc/&#176;. 
		NOTE: For squirrels, we devised a protocol where we perform the acuity test but at a much lower contrast. We selected a contrast setting (12.78%) at which the squirrels were unable to see the striped pattern at 2 cycles/&#176;.</li>
	</ol>
	</li>
	<li>Download Supplementary Coding File 3. Open the OptoDrum software.</li>
	<li>Under the Settings tab, click on Import Settings. Load the Supplementary Coding File 3.</li>
	<li>Under the Settings tab, set the Background threshold offset to 17, the minimum animal size [px] to 30, and the maximum tail width [% of size] to 1.</li>
	<li>Under camera settings, leave Invert Video and Manual Camera control unchecked and IR Light Off.</li>
	<li>Widen the position and size of the region of interest for tracking to accommodate the squirrel if it leans outside of the opening of the tube. The suggested values are Position: X: 359, Y: 237, Size: X: 570, Y: 455. Do not extend the ROI over the Stimuli screen, as it can lead to false positives.</li>
	<li>Select Session Configuration from the top panel (Figure 2A).
	<ol>
		<li>For the staircase, set estimated spatial acuity as follows. Set expected acuity to 1.800 cyc/&#176; (648 cycles); set optimal stimulus resolution to 0.5 cyc/&#176; (180 cycles); set measurement resolution to 0.100 cyc/&#176; (36 cycles).</li>
		<li>For the staircase, set the number of required confirmations as follows. Set for failed trials to 3; set for successful trials to 2. 
		NOTE: The number of successful trials can be increased to further reduce the occurrence of false positive confirmations. However, with the use of this platform and modified handling tunnel, the error rate remains acceptably low. This is primarily due to the squirrels&#39; relative stillness and the infrequency of rotational head movements aside from those elicited by visually evoked stimuli.</li>
	</ol>
	</li>
	<li>Under the Staircase tab (Figure 2B), set antialiasing to width to 3 px and leave sinusoidal unchecked.</li>
	<li>For the testing criteria, lock the Contrast at 12.78% and lock the rotation speed at 12 &#176;/s. 
	NOTE: At the 12.78% contrast setting, the squirrels&#39; spatial acuity is limited to approximately 1.5-1.8 cyc/&#176;. No longer exceeding the maximum hardware settings, this configuration enables the assessment of spatial acuity in response to disease or injury.</li>
	<li>Once two of the three parameters are locked, the Auto Set the parameters option will become active (no longer grayed out). Check the box next to Auto Set the Parameters.</li>
	<li>Set the rotation direction depending on whether visual impairment is expected to be in one or both eyes. Note that for each eye, only motion in the temporal-to-nasal direction elicits tracking. Consequently, assess injury to the left eye using clockwise (CW) stimulation, as counterclockwise (CCW) stimulation is expected to elicit normal responses. For degeneration or injury expected in both eyes, rotation of the stimulus in both directions may be used.</li>
	<li>Click on Start Trial to begin the presentation of the stimulus. The camera above will begin recording and tracking the animals&#39; head movements. While the stimulus is being presented, click on the Analysis Tab for real-time viewing of the Angular Velocities (head rotation), Track Quality, and Score parameters. At the end of each stimulus presentation, the software will automatically determine whether to advance (indicated by the green checkmark) to the next Cyc/&#176;, repeat the trial, or reduce the Cyc/&#176; (indicated by the red X).</li>
	<li>During the recording, if the tracking software incorrectly flips the nose (red) and tail (green) markers, press Ctrl on the keyboard to allow manual override to correct the head-tail orientation.</li>
	<li>If the animal falls from the platform, pause the trial by pressing Pause Evaluation or clicking on the Space Bar. Remove the handling tunnel from the platform and use it with the lid for safe handling of the animal to place it back onto the platform. Once the animal is ready, click on Resume Evaluation to continue the trial.</li>
	<li>Once all the trials have been completed, click on the Summary Tab to show the successful trials in green and the unsuccessful trials in red. The trial circled in green indicates the visual acuity threshold for the squirrel using the contrast of 12.78%. Return the animal carefully to its home cage.</li>
</ol>

<ol><li>Ferguson, R., Miyagishima, K. J., Nadal-Nicolas, F. M., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+the+rod+pathway+in+cone-dominated+thirteen-lined+ground+squirrels%5BTitle%5D)+AND+%22Front+Ophthalmol%22%5BJournal%5D)">Characterizing the rod pathway in cone-dominated thirteen-lined ground squirrels.</a> Front Ophthalmol. 3, 1271882(2023).</li>
<li>Nadal-Nicolas, F. M., Miyagishima, K. J., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+search+for+the+%26%2334%3Bidyllic%26%2334%3B+animal+model+to+evaluate+ocular+pathologies+and+translate+new+therapies+to+improve+human+health%5BTitle%5D)+AND+%22Neural+Regen+Res%22%5BJournal%5D)">In search for the &#34;idyllic&#34; animal model to evaluate ocular pathologies and translate new therapies to improve human health.</a> Neural Regen Res. 17 (12), 2697-2699 (2022).</li>
<li>Xiao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishing+the+ground+squirrel+as+a+superb+model+for+retinal+ganglion+cell+disorders+and+optic+neuropathies%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">Establishing the ground squirrel as a superb model for retinal ganglion cell disorders and optic neuropathies.</a> Lab Invest. 101 (9), 1289-1303 (2021).</li>
<li>Yanoga, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sildenafil+citrate+induced+retinal+toxicity-electroretinogram%2C+optical+coherence+tomography%2C+and+adaptive+optics+findings%5BTitle%5D)+AND+%22Retin+Cases+Brief+Rep%22%5BJournal%5D)">Sildenafil citrate induced retinal toxicity-electroretinogram, optical coherence tomography, and adaptive optics findings.</a> Retin Cases Brief Rep. 12 (Suppl 1), S33-S40 (2018).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug-induced+colour+vision+disorders%5BTitle%5D)+AND+%22Prescrire+Int%22%5BJournal%5D)">Drug-induced colour vision disorders.</a> Prescrire Int. 21 (127), 126-128 (2012).</li>
<li>Widengard, I., Mandahl, A., Tornquist, P., Wistrand, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Colour+vision+and+side-effects+during+treatment+with+methazolamide%5BTitle%5D)+AND+%22Eye%22%5BJournal%5D)">Colour vision and side-effects during treatment with methazolamide.</a> Eye. 9 (Pt 1), 130-135 (1995).</li>
<li>Benkner, B., Mutter, M., Ecke, G., Munch, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+visual+performance+in+mice%3A+An+objective+and+automated+system+based+on+the+optokinetic+reflex%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">Characterizing visual performance in mice: An objective and automated system based on the optokinetic reflex.</a> Behav Neurosci. 127 (5), 788-796 (2013).</li>
<li>Chang, K., Chen, J., Rajagopalan, A., Chen, D. F., Cho, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testing+visual+function+by+assessment+of+the+optomotor+reflex+in+glaucoma%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Testing visual function by assessment of the optomotor reflex in glaucoma.</a> Methods Mol Biol. 2858, 219-227 (2025).</li>
<li>Mui, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Daily+visual+stimulation+in+the+critical+period+enhances+multiple+aspects+of+vision+through+bdnf-mediated+pathways+in+the+mouse+retina%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Daily visual stimulation in the critical period enhances multiple aspects of vision through bdnf-mediated pathways in the mouse retina.</a> PLoS One. 13 (2), e0192435(2018).</li>
<li>Prusky, G. T., Alam, N. M., Beekman, S., Douglas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+quantification+of+adult+and+developing+mouse+spatial+vision+using+a+virtual+optomotor+system%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system.</a> Invest Ophthalmol Vis Sci. 45 (12), 4611-4616 (2004).</li>
<li>Shi, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+optomotor+response-based+visual+function+assessment+in+mice%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Optimization of optomotor response-based visual function assessment in mice.</a> Sci Rep. 8 (1), 9708(2018).</li>
<li>Kretschmer, F., Tariq, M., Chatila, W., Wu, B., Badea, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+optomotor+and+optokinetic+reflexes+in+mice%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Comparison of optomotor and optokinetic reflexes in mice.</a> J Neurophysiol. 118 (1), 300-316 (2017).</li>
<li>Kretschmer, F., Kretschmer, V., Kunze, V. P., Kretzberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Omr-arena%3A+Automated+measurement+and+stimulation+system+to+determine+mouse+visual+thresholds+based+on+optomotor+responses%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Omr-arena: Automated measurement and stimulation system to determine mouse visual thresholds based on optomotor responses.</a> PLoS One. 8 (11), e78058(2013).</li>
<li>Kretschmer, F., Sajgo, S., Kretschmer, V., Badea, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+system+to+measure+the+optokinetic+and+optomotor+response+in+mice%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A system to measure the optokinetic and optomotor response in mice.</a> J Neurosci Methods. 256, 91-105 (2015).</li>
<li>Abdeljalil, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+optomotor+response%3A+A+robust+first-line+visual+screening+method+for+mice%5BTitle%5D)+AND+%22Vision+Res%22%5BJournal%5D)">The optomotor response: A robust first-line visual screening method for mice.</a> Vision Res. 45 (11), 1439-1446 (2005).</li>
<li>Thaung, C., Arnold, K., Jackson, I. J., Coffey, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+visual+head+tracking+differentiates+normal+sighted+from+retinal+degenerate+mice%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">Presence of visual head tracking differentiates normal sighted from retinal degenerate mice.</a> Neurosci Lett. 325 (1), 21-24 (2002).</li>
<li>Jecrois, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+during+a+developmental+window+prevents+nf1-associated+optic+pathway+gliomas+by+targeting+erk-dependent+migrating+glial+progenitors%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Treatment during a developmental window prevents nf1-associated optic pathway gliomas by targeting erk-dependent migrating glial progenitors.</a> Dev Cell. 56 (20), 2871-2885.e6 (2021).</li>
<li>Kandoi, S., Martinez, C., Merriman, D. K., Lamba, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+retinal+development+in+13-lined+ground+squirrels%5BTitle%5D)+AND+%22Transl+Vis+Sci+Technol%22%5BJournal%5D)">Characterization of retinal development in 13-lined ground squirrels.</a> Transl Vis Sci Technol. 11 (11), 17(2022).</li>
<li>Kryger, Z., Galli-Resta, L., Jacobs, G. H., Reese, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+topography+of+rod+and+cone+photoreceptors+in+the+retina+of+the+ground+squirrel%5BTitle%5D)+AND+%22Vis+Neurosci%22%5BJournal%5D)">The topography of rod and cone photoreceptors in the retina of the ground squirrel.</a> Vis Neurosci. 15 (4), 685-691 (1998).</li>
<li>Savignac, H. M., Dinan, T. G., Cryan, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resistance+to+early-life+stress+in+mice%3A+Effects+of+genetic+background+and+stress+duration%5BTitle%5D)+AND+%22Front+Behav+Neurosci%22%5BJournal%5D)">Resistance to early-life stress in mice: Effects of genetic background and stress duration.</a> Front Behav Neurosci. 5, 13(2011).</li>
<li>Hulliger, E. C., Hostettler, S. M., Kleinlogel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Empowering+retinal+gene+therapy+with+a+specific+promoter+for+human+rod+and+cone+on-bipolar+cells%5BTitle%5D)+AND+%22Mol+Ther+Methods+Clin+Dev%22%5BJournal%5D)">Empowering retinal gene therapy with a specific promoter for human rod and cone on-bipolar cells.</a> Mol Ther Methods Clin Dev. 17, 505-519 (2020).</li>
<li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+phagocytosis+of+living+photoreceptors+contributes+to+inherited+retinal+degeneration%5BTitle%5D)+AND+%22EMBO+Mol+Med%22%5BJournal%5D)">Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration.</a> EMBO Mol Med. 7 (9), 1179-1197 (2015).</li>
<li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pde6+mutation+in+the+rd10+retinal+degeneration+mouse+model+causes+protein+mislocalization+and+instability+and+promotes+cell+death+through+increased+ion+influx%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">The pde6 mutation in the rd10 retinal degeneration mouse model causes protein mislocalization and instability and promotes cell death through increased ion influx.</a> J Biol Chem. 293 (40), 15332-15346 (2018).</li>
<li>Hwang, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythm+of+contrast+sensitivity+is+regulated+by+a+dopamine-neuronal+pas-domain+protein+2-adenylyl+cyclase+1+signaling+pathway+in+retinal+ganglion+cells%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Circadian rhythm of contrast sensitivity is regulated by a dopamine-neuronal pas-domain protein 2-adenylyl cyclase 1 signaling pathway in retinal ganglion cells.</a> J Neurosci. 33 (38), 14989-14997 (2013).</li>
<li>Merriman, D. K., Lahvis, G., Jooss, M., Gesicki, J. A., Schill, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+practices+in+a+captive+breeding+colony+of+13-lined+ground+squirrels+%28Ictidomys+tridecemlineatus%29%5BTitle%5D)+AND+%22Lab+Anim%22%5BJournal%5D)">Current practices in a captive breeding colony of 13-lined ground squirrels (Ictidomys tridecemlineatus).</a> Lab Anim. 41 (11), 315-325 (2012).</li>
<li>Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ground+squirrel+-+a+cool+model+for+a+bright+vision%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Ground squirrel - a cool model for a bright vision.</a> Semin Cell Dev Biol. 106, 127-134 (2020).</li>
<li>Gudapati, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+assessment+of+visual+function+via+optomotor+response+and+cognitive+function+via+y-maze+in+diabetic+rats%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Behavioral assessment of visual function via optomotor response and cognitive function via y-maze in diabetic rats.</a> J Vis Exp. (164), e61806(2020).</li>
<li>Stojcev, M., Radtke, N., D'amaro, D., Dyer, A. G., Neumeyer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((General+principles+in+motion+vision%3A+Color+blindness+of+object+motion+depends+on+pattern+velocity+in+honeybee+and+goldfish%5BTitle%5D)+AND+%22Vis+Neurosci%22%5BJournal%5D)">General principles in motion vision: Color blindness of object motion depends on pattern velocity in honeybee and goldfish.</a> Vis Neurosci. 28 (4), 361-370 (2011).</li>
<li>Sajdak, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+imaging+of+the+tree+shrew+eye%3A+Wavefront+analysis+and+retinal+imaging+with+correlative+histology%5BTitle%5D)+AND+%22Exp+Eye+Res%22%5BJournal%5D)">Noninvasive imaging of the tree shrew eye: Wavefront analysis and retinal imaging with correlative histology.</a> Exp Eye Res. 185, 107683(2019).</li>
<li>Okawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+processing+of+photoreceptor+signals+is+required+to+maximize+behavioural+sensitivity%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Optimal processing of photoreceptor signals is required to maximize behavioural sensitivity.</a> J Physiol. 588 (Pt 11), 1947-1960 (2010).</li>
</ol>

Kiyoharu Miyagishima, Francisco Nadal-Nicol&#225;s, John Ball, and Wei Li declare no competing financial interests. Thomas Munch is the Owner and Director of Research and Development at Striatech GmbH. Boris Benkner is the Founder and Chief Executive Officer at Striatech GmbH.

Critical steps
Facilitating Tunnel Entry - One important point to consider is that the squirrels require the 3D-printed enclosure assembly to serve as a temporary lid for the acrylic rectangular tunnel; without it, they will not readily enter. In our facility, the 13-lined ground squirrels are regularly housed with a red rat-sized cylinder (Length: 15.24 cm, Diameter: 7.62 cm). The purpose of the 3D-printed enclosure is to provide familiarity with their enrichment tunnels, mak...

Ground squirrels offer a unique and valuable model for studying visual function due to their diurnal lifestyle and cone-dominated retinas, which closely mimic human vision1. This, coupled with the practical advantages of their use (smaller size, lower husbandry cost) compared to primates, make them an ideal model for translational research into retinal injury, disease, or ocular side effects of drugs on color vision changes2,3,4,5,6. Preclinical research on retinal diseases relies on the ability to assess whether an animal can see. The optomotor reflex (OMR) offers a clinically relevant method for evaluating visual function in animals without the need for anesthesia, enabling an accurate measurement of visual acuity7,8. In this test, the animal follows a rotating stripe pattern with its gaze. The rotating stripes induce the perception of global motion in the environment, triggering an involuntary head movement - the OMR. This reflex serves to visually stabilize the environment and can be used to quantify how well an animal can see by incrementally adjusting the spatial frequency or contrast of the stimulus. As the stimulus becomes more difficult to perceive, the OMR is eventually not triggered, allowing the determination of visual acuity or contrast threshold. The advantage of the OMR is that it is a reflex. Thus, no training is required, and head fixation or surgery is unnecessary. The reflex is present even in very young animals (upon eye-opening)9,10, making it possible to study early-onset diseases. Additionally, since it is noninvasive, longitudinal measurements can be made enabling tracking of disease progression.
Most OMR testing is performed using custom or commercial systems that consist of a raised platform where the animal is placed, surrounded by four screens displaying rotating black and white stripe patterns. The stripes move in one direction (clockwise or counterclockwise), creating a motion stimulus that elicits a visual tracking response from the animal10,11,12,13,14,15,16. A camera is mounted above the testing arena to capture the animal&#39;s visually evoked head movements in response to the rotating stimulus. These head movements are tracked and analyzed to determine the animal&#39;s ability to perceive and respond to motion, thereby assessing its visual acuity.
Although the OMR is routinely used in mice and rats, measurement of the optomotor response in 13-lined ground squirrels has been challenging due to their hyperactivity and inability to stay on elevated platforms, even with prior conditioning. To circumvent this, we developed a platform that provides semi-containment, facilitating the transfer of the animals to the arena. The goal of this method is to reduce the time for the animals to adapt to the experimental setting, enabling the recording of animals that display uncooperative behavior or have difficulty staying on the platform and improving the inclusion of animals in studies. We have also defined a protocol to measure visual acuity in squirrels using an experimentally defined contrast setting and have adjusted the camera settings for improved imaging results, enabling automated analysis and tracking.

<table><tbody><tr><td>12V Max Lithium-ion Cordless Rotary Tool Kit</td><td>Dremel 8220</td><td>F0138220JA</td><td>To cut and polish the Acrylic Square Tube.&lt;br/&gt; https://www.dremel.com/us/en/p/8220-1-28-f0138220aa</td></tr><tr><td>6-0 COATED VICRYL (polyglactin 910) Suture</td><td>Ethicon</td><td>J670G</td><td>For suturing the eye lid.&lt;br/&gt; https://www.ethicon.com/na/epc/code/j670g?lang=en-default</td></tr><tr><td>Clear Colorless Acrylic Square Tube (2&quot; x 12&quot;)</td><td>Canal Plastics Center</td><td>RT-300161</td><td>Clear rectangular tunnel (for mouse).&lt;br/&gt; https://www.canalplastic.com/products/clear-colorless-acrylic-square-tube</td></tr><tr><td>Clear Colorless Acrylic Square Tube (3&quot; x 12&quot;)</td><td>Canal Plastics Center</td><td>RT-300168</td><td>Clear rectangular tunnel (for squirrel).&lt;br/&gt; https://www.canalplastic.com/products/clear-colorless-acrylic-square-tube</td></tr><tr><td>DREMEL EZ SpeedClic: Plastic Cutting Wheels</td><td>Dremel</td><td>SC476</td><td>For cutting the Acrylic Square Tube</td></tr><tr><td>F270 3D printer</td><td>Stratasys</td><td></td><td>To print engineering grade 3D models of the platforms.&lt;br/&gt; https://support.stratasys.com/en/printers/fdm-legacy/uprint</td></tr><tr><td>Fluriso (Isoflurane)</td><td>VetOne&amp;nbsp;</td><td>502017</td><td>General Anesthesia. Liquid for Inhalation.&lt;br/&gt; http://vetone.net/Default/GetFile?id=7dda0c60-45fa-e711-bf2e-0024e8785118</td></tr><tr><td>FreeCAD 1.0&amp;nbsp;</td><td></td><td></td><td>Open-source parametric 3D modeler</td></tr><tr><td>Igor Pro software Version 6.3.7.2</td><td>WaveMetrics, Inc</td><td></td><td>For plotting the graphs. https://www.wavemetrics.com/</td></tr><tr><td>Low-Profile Anesthesia Masks for Traditional Vaporizers&lt;br/&gt; &amp;nbsp;for SomnoFlo. Extra Large (animals over 300 g)&amp;nbsp;</td><td>Kent Scientific corporation</td><td>SOMNO-0804</td><td>For providing general anesthesia.&lt;br/&gt; https://www.kentscientific.com/products/low-profile-anesthesia-masks-for-somnosuite/</td></tr><tr><td>MakerBot Print for Windows version 4.10.1.2056</td><td>MakerBot Industries, LLC</td><td></td><td>Software for orienting the parts for printing</td></tr><tr><td>MakerBot Replicator Desktop 3D Printer (5th Gen)</td><td>MakerBot Industries, LLC</td><td>MP05825</td><td>For 3D printing prototype versions of the platforms.&lt;br/&gt; https://www.rnd-tech.com/product/makerbot-replicator-desktop-3d-printer-5th-gen/</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td>Spreadsheet software to analyze the data. microsoft.com/en-us/microsoft-365/excel</td></tr><tr><td>Multipurpose leather work gloves</td><td>Steiner Industries</td><td>SPC02</td><td>To handle squirrels.&lt;br/&gt; https://www.steinerindustries.com/leather-palm/product/steiner-spc02-leather-palm-work-glove</td></tr><tr><td>OptoDrum Plus</td><td>STRIA.TECH</td><td></td><td>Commercial device for measuring the optomotor reflex (visual acuity and contrast sensitivity) https://stria.tech/products/optodrum/&amp;nbsp;</td></tr><tr><td>OptoDrum software Version 1.7.3</td><td>STRIA.TECH</td><td></td><td>Software for performing automated measures of the optomotor reflex.</td></tr><tr><td>Peroxigard Ready to Use (cleaner/disinfectant)&amp;nbsp;</td><td>Peroxigard</td><td>PRTU242101</td><td>To clean/disinfect the platform and the acrylid square tube between experiments.&lt;br/&gt; https://peroxigard.com/product-information/rtu/</td></tr><tr><td>Pureline M6000 Anesthesia Machine&lt;br/&gt; with O2 Concentrator</td><td>Penn Veterinary Supply, Inc.</td><td>SUPM6000</td><td>For animal anesthesia.&lt;br/&gt; https://www.pennvet.com/customer/portal/catalog/home?urile=wcm:path%3APennVet+Catalog/Product+Catalog/SUPM6000/Pureline+M6000+Anesthesia+Mach+w_O2+Conc</td></tr><tr><td>uPrint SE Plus 3D printer</td><td>Stratasys</td><td></td><td>To print engineering grade 3D models of the platforms.&lt;br/&gt; https://www.makerbot.com/makerbot-print/</td></tr><tr><td>White opaque MakerBot PLA Filament,&lt;br/&gt; Large Spool for Replicator+ (0.9kg, 2lb)</td><td>MakerBot Industries, LLC</td><td>MP05780</td><td>Filament to 3D print the platforms.&lt;br/&gt; https://store.ultimaker.com/3d-printer-materials/replicator-series-materials/makerbot-pla-material-large-spool-for-replicator</td></tr><tr><td>Pharmacological treatment</td><td></td><td></td><td></td></tr><tr><td>Neomycin and Polymyxin B Sulfates&lt;br/&gt; and Bacitracin Zinc Ophthalmic Ointment, USP</td><td>Bausch &amp;amp; Lomb</td><td>RX-0069</td><td>Triple ophthalmic antibiotic for preventing ocular infections</td></tr><tr><td>Proparacaine Hydrochloride 0.5%</td><td>Akorn</td><td>17478-263-12</td><td>Local anesthetic for ophthalmic instillation</td></tr></tbody></table>

non-aversive animal restraint enabling recording of optomotor reflex in ground squirrels

Non-aversive Animal Restraint Enabling Recording of Optomotor Reflex in Ground Squirrels

The optomotor reflex (OMR) provides a behavioral assessment of an animal's contrast sensitivity and visual acuity. Mice or rats are typically placed directly onto a small circular platform by hand; however, handling animals like this can stimulate stress and anxiety, which introduce confounding factors when interpreting data. It has been shown that non-aversive handling methods, such as picking up mice or rats in a familiar tunnel/tube, can reduce anxiety. This is of particular interest in studies where animals display heightened stress, overactivity, or motor dysfunction, resulting in an inability to stay on the platform. A team led by Drs. Kiyoharu J. Miyagishima and Francisco M. Nadal-Nicol&#225;s have redesigned the conventional OMR platform to provide semi-closed containment. This makes it possible for the first time to record the optomotor reflex in the 13-lined ground squirrel, which is one of the few mammals that can see color. It has a visual streak with a high density of cones similar to the human macula providing an attractive model for studying effects on the cone visual system.

OMR is considered successful once the software identifies the visual threshold determined by the animal's performance in spatial frequency discrimination trials. Specifically, the threshold is defined as follows: two successful trials at a given spatial frequency (measured in cyc/°), followed by three failed trials at the next higher spatial frequency. OMR was performed on 13-lined ground squirrels (6-8 months of age) using the standard round flat platform (Figure 3A) to compare with the non...

The protocol describes how to measure the optomotor response in 13-lined ground squirrels, a cone-dominant species with retinal features resembling the human macula. The platform design enhances handling and reduces anxiety, improving the animal's ability to stay on the raised platform, enabling non-invasive assessment of changes in visual function.

The optomotor reflex (OMR) provides a behavioral assessment of an animal's contrast sensitivity and visual acuity. Mice or rats are typically placed ...

non-aversive-animal-restraint-enabling-recording-optomotor-reflex

Research

JoVE Journal

Behavior

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Video-oculography in Mice

This video demonstrates how to record electroretinogram (ERG) and visual evoked potential (VEP) in a conscious rat. The rat, with surgically implanted electrodes, is first dark-adapted. After applying dilating drops, the animal is placed in a restrainer and positioned in a Ganzfeld bowl. The ERG and VEP signals are then recorded.

Recording of Electroretinogram and Visual Evoked Potential in a Rat

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats.  It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term Sensory Conflict in Freely Behaving Mice

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360° thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

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.

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

Learn to measure optomotor responses in 13-lined ground squirrels using a non-aversive platform design. This method reduces anxiety and enables reliable visual function assessment without extensive acclimatization.

Non-aversive Animal Restraint Enabling Recording of Optomotor Reflex in Ground Squirrels

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