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

The OMR is widely used to screen visual impairments, but has been limited to traditional models like mice and rats. Our designed platform enables recordings from 13 line ground squirrels, which have a cone rich visual streak resembling the human macula.

Repeated escape atam or inattentive exploratory behavior can disrupt recording, compromising accuracy and limiting sample sizes.

Combining the platform with a familiar transfer like tube promotes quick habituation, reduces anxiety like behaviors, minimizes stress, and lowers the risk of falls.

Ground squirrel often struggle with a stability on a standard round platform, but the new semi enclosed design improve engagement and enable renewable data collection without extensive acclimatization or positive reinforcement.

The ability to record from cone dominant mammals opens new avenues to investigate drug-induced ocular side effects, including changes in color vision.

[Narrator] To begin wipe the platform and the square tube enclosure assembly using a cleaner and disinfectant to eliminate unwanted olfactory cues, tear a piece of paper towel approximately 50 by 50 millimeters and fold it to cover the top surface of the pedestal. Place the 3D printed platform onto the 14 inch high pedestal with the folded paper towel in place. Wearing leather work gloves, gently guide the animal into the square tube enclosure assembly. Transfer the animal to the arena by positioning the enclosure assembly onto the platform. Now carefully detach the removable top enclosure, leaving the animal inside the acrylic extruded clear square tube on the platform. Close the door to the optomotor reflex system. Typically, visual acuity tests on rodents are performed using the standard 100% contrast setting. However, squirrels can see well beyond the spatial frequency of mice and rats, even beyond the limits of the software, which is a maximum of two cycles per degree. At two cycles per degree, each stripe averages 5.3 pixels wide. Now set the rotation speed to 12 degrees per second. After importing the squirrel track file, go to the settings tab, confirm the background threshold offset is set to 17, the minimum animal size is set to 30 pixels, and the maximum tail width to 1% of size. Verify that the animal's features are tracked correctly in the video panel. If not, adjust the offset incrementally until tracking is accurate. Under camera settings, ensure that invert video and manual camera control are unchecked and IR light is off. Widen the region of interest to accommodate the squirrel if it leans outside the tube opening. Set position to X 359, Y 237, and size to X 570, Y 455. Next select session configuration from the top panel. Under the staircase section, set the estimated spatial acuity values. Enter 1.8 cycles per degree for expected acuity, 0.5 cycles per degree for optimal stimulus resolution and 0.1 cycles per degree for measurement resolution. Then set the number of required confirmations in the staircase settings. Enter three confirmations for failed trials and two for successful trials. For testing criteria, lock the contrast at 12.78% and the rotation speed at 12 degrees per second. Once two of the three parameters are locked, check the box next to auto set the parameters once it becomes active, then set the rotation direction based on the expected location of visual impairment. Use clockwise stimulation to assess the left eye or use both clockwise and counterclockwise stimulation for bilateral assessment. Now click on start trial to begin stimulus presentation. As the stimulus plays, click on the analysis tab to monitor angular velocities, track quality, and score in real time. At the end of each trial, the software will automatically indicate whether to advance, repeat, or reduce the cycle per degree based on performance. If the software incorrectly switches the red nose and green tail markers during tracking, press control to activate manual override and correct the head tail orientation. If the animal leaves the platform, pause the trial with the pause evaluation option or the space bar. Use the handling tunnel with the lid to safely return the animal to the platform and click on resume evaluation to continue the trial. After all trials are complete, click on the summary tab to view results. Successful trials will appear in green and unsuccessful ones in red. The green circle trial indicates the visual acuity threshold. Finally return the animal to its home cage carefully. Animals placed on the standard round flat platform for the OMR test invariably fell off and were unable to complete the automated visual threshold assessment within the allotted 10 minutes. In contrast, animals placed on the non aversive platform with the handling tunnel completed OMR measurements successfully in all cases without prior acclimation or reward conditioning. The modified tunnel allowed the animal to reorient and view stimuli from any direction enhancing natural behavior during testing. The tracking software successfully monitored head movement as the animal responded to directional stimuli. Overlaying frames taken two seconds apart, showed reflexive head tracking in response to visual stimuli. The software's real-time analysis differentiated visually evoked from random head movements, correlating with the stimulus presentation windows. Using the staircase method, the software quickly determined visual thresholds with most stimuli requiring less than 30 seconds of presentation.