Name: Using Looming Visual Stimuli to Evaluate Mouse Vision (Video) | JoVE Journal
Uploaded: 2019-06-13T14:30:00.000+00:00
Duration: 5 min 7 s
Description: Learn looming visual stimuli testing for mouse vision using behavioral assays. This robust method evaluates image forming pathways through freezing and flight responses without extensive training.

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5 minutes

This work was supported by NIH R01 EY028915 (TI) and RPB grants.

All experiments and animal care were conducted in accordance with protocol approved by the Institutional Animal Care and Use Committees at Wayne State University (protocol no. 17-11-0399).
1. Preparation for the experiment
<ol>
	<li>Build a rectangular open-lid enclosure to house the mouse during looming visual stimuli presentation. We constructed a 40 cm x 50 cm x 33 cm enclosure using aluminum framing and PVC panels (Figure 1A,B). Lay a sheet of paper to cover the entire floor of the enclosure to ensure easy cleanup between trials. Add an opaque shelter in a corner of the enclosure with an entrance facing the center of the arena for easy entrance and exit.</li>
	<li>Set up a camera with a wide-angle lens for capturing the mouse&#8217;s behavior. Secure the camera to a table-mounted stand adjacent to the enclosure. For best quality video capturing, use a camera frame rate of 60 FPS or higher.</li>
	<li>Set up a computer monitor on top of the enclosure. Because the monitor could not be seen from the outside, a second monitor was prepared, which duplicated the images shown on the primary monitor.</li>
	<li>Prepare a looming pattern for projection. One way to do this is to use the PsychToolbox3 within MatLab software to code for an expanding black circle (Figure 1C). Set the stimulus to begin at a visual angle of 2&#176; and expand to 50&#176; over 250 ms; these parameters determine stimulus speed (see Figure 1D for visual angle calculation). Set the code to repeat the stimulus 10 times with an interval of 1 s. 
	NOTE: The stimulus began each repetition immediately upon termination of the previous presentation. For further information on stimulus presentation, please refer to section 3.</li>
	<li>Select mice of interest for the looming stimuli. Here, use 32 healthy-eyed mice of a C57 background, male and female, 4 to 14 weeks old. Also, use 3 blind mice (severe cataracts in both eyes) to assess whether the response to looming stimulus was truly a visually guided behavior. These blind mice had no pupillary light reflex and no optomotor response.</li>
</ol>
2. Mouse acclimation
<ol>
	<li>Place a mouse in the enclosure and let it freely explore its surroundings. If possible, try to minimize stress during animal transfer by using the back of your free hand as a resting place for the mouse instead of letting it hang without support. The mouse should find the entire enclosure to be safe and should discover the hiding place. Drop a few food pellets in the corner opposite the refuge to encourage the mouse to remain outside the refuge.</li>
	<li>Allow the mouse to acclimate anywhere from 7 to 15 min29,39. We allowed 10 min of acclimation prior to stimulus onset. Furthermore, 10 min acclimation one day prior to the experiment may ease the mice.</li>
</ol>
3. Looming visual stimuli projection
<ol>
	<li>Prior to inserting the mouse into the arena, make sure the stimulus code is ready to run to facilitate as few lighting changes as possible while the mouse is in the enclosure. Once the software is ready to run, gently place the mouse in the enclosure.</li>
	<li>10 seconds prior to the stimulation, start the video capturing.</li>
	<li>Start the looming visual stimuli when the mouse is away from the shelter and moving freely in the open arena. Wait 10 seconds after the last stimulus presentation to terminate the recording.
	<ol>
		<li>Begin the stimulus presentation when the mouse is in the corner farthest from the refuge. However, when mice seem unwilling to explore the far corner, present the stimulus when the mouse is in a different corner of the arena. This does not appear to make a difference in animal behavioral response.</li>
	</ol>
	</li>
	<li>Transfer the mouse back to its original cage. Clean the enclosure for the next mouse by spraying the walls and refuge with 70% ethanol and wiping it down. Replace the paper floor liner if soiled and reposition the refuge to the same initial location if moved during animal transfer and enclosure cleaning.</li>
</ol>
4. Video analysis
<ol>
	<li>Save the video clip for each mouse in .avi format without file compression in order to ensure no data loss during transfer to the analysis software. 
	NOTE: Lack of compression will lead to larger file size; therefore, use external hard drive for storage.</li>
	<li>Use analysis software to track the animal&#8217;s motion around the arena prior to, during, and after stimulus presentation. Use commercially available software (see Table of Materials) with a manual tracking ability to track the position of the mouse head in every video frame, which generated X and Y coordination every 1/60 ms. Other motion-tracking software includes FIJI (NIH)40 and EthoVision (Noldus).</li>
	<li>Calculate the velocity and the distance of the mouse from the refuge. If the image of the arena is distorted due to the video angle, correct the X and Y coordination prior to the calculation (Figure 2).</li>
	<li>Compare the parameters before and after looming stimulus onset to determine how the mouse responded to the stimuli, whether by freezing, fleeing, or demonstrating no change in behavior29. Define freezing as episodes where speed was less than 20 mm/s for 0.5 s or longer. Define flight as episodes where speed increased to 400 mm/s or greater and ended with the mouse in the refuge. Definitions for freezing and flight were based on those set by Franceschi et al.29</li>
</ol>

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</ol>

The authors have nothing to disclose.

With the looming visual stimuli system, a majority (97%) of healthy eye-mice showed flight response. One of 29 mice did not show an obvious flight response. However, the mouse walked toward the dome and remained near it until looming disappeared, indicating that the mouse was at least cautious when the looming stimuli occurred. Therefore, the looming stimuli consistently elicited innate fear responses in healthy-eyed mice. On the other hand, three blind mice did not show any responses to the looming (preliminary results)...

The visual system starts at the retina, where visual signals are captured by photoreceptors, channeled to bipolar cells (2nd-order neurons), and finally passed to ganglion cells (3rd-order neurons). Retinal 2nd- and 3rd-order neurons are thought to form multiple neural pathways that convey particular aspects of visual signaling such as color, motion, or shape. These diverse visual features are relayed to the lateral geniculate nucleus and the visual cortex. In contrast, visual signals leading to eye movement are sent to the superior colliculus. Classically, two retino-cortical pathways have been identified: the magnocellular and the parvocellular pathways. These pathways encode moving and stationary objects, respectively, and their existence embodies the basic concept of parallel processing1,2,3,4,5,6. Recently, more than 15 types of bipolar cells7,8,9,10,11 and ganglion cells12,13,14,15,16 have been reported in the retina of many species, including the primate retina. These cells are distinguished not only by morphological aspects, but also by the expression of distinct markers and genes8,10,17,18, suggesting that various features of visual signals are processed in parallel, which is more complicated than originally anticipated.
Cellular and molecular technologies have contributed to our understanding of visual processing and potential disease mechanisms that may arise from aberrant visual processing. Such an understanding may contribute to the development of artificial eyes. Although cellular examinations and analysis offer in-depth knowledge at a cellular level, a combination of behavioral experiments and cellular experiments would significantly augment our current understanding of minute visual processes. For example, Yoshida et al.19 found that starburst amacrine cells are the key neurons for motion detection in the mouse retina. Following cellular experiments, they performed the optokinetic nystagmus (OKN) behavioral experiment to show that mutant mice in which starburst amacrine cells were dysfunctional did not respond to moving objects, thereby confirming their cellular investigations. In addition, Pearson et al.20 conducted photoreceptor transplantation in the mouse retina to restore vision in diseased mice. They conducted not only cellular experiments, but also measured mouse behavior through the use of optomotor response recordings and water-maze tasks thus allowing Pearson et al. to verify that transplanted photoreceptors restored vision in the formerly blind mice. Taken together, behavioral experiments are strong tools to assess mouse vision.
Multiple methods are available for measuring mouse vision. These methods have advantages and limitations. In vivo ERG provides information on whether the mouse retina, particularly photoreceptors and ON bipolar cells, appropriately responds to light stimuli. ERG can be tested either under scotopic or photopic conditions21,22. However, ERG requires anesthesia, which might affect the output measurement23. The optokinetic reflex (OKR) or optomotor response (OMR) is a robust method to assess contrast sensitivity and spatial resolution, both functional components of mouse vision. However, OKR requires surgery to attach a fixation device to the mouse skull24. OMR requires neither surgery nor mouse training; however, it requires training to allow an experimenter to subjectively detect subtle mouse head movements in response to a moving grating in an optic drum 25,26. Pupil light reflex measures pupil constriction in response to light stimuli, which does not require anesthesia and exhibits objective and robust responses 19. Although the pupil reflex simulates retinal light response in vivo, the reflex is mediated mainly by the intrinsically photosensitive retinal ganglion cells (ipRGCs) 27. Because ipRGCs represent a small minority of RGCs and do not serve as conventional image-forming ganglion cells, this measurement does not provide information pertaining to the majority of ganglion cells.
The looming light experiment has not previously been considered a major test for measuring mouse vision. However, it is also a robust and reliable vision test across various species, such as mouse28,29, zebrafish30, locust31,32, and human33,34,35. Importantly, the looming experiment is one of only a few methods to test the image-forming pathway - it is not a reflex pathway - given the visual and the limbic systems in the central nervous system are involved in this circuit36,37,38. We have established a looming visual stimulus system and have demonstrated its ability to elicit motion detection in the mouse, which we use as a proxy to assess the intactness of the mouse visual system.

<table><tbody><tr><td>10.1&quot; monitor (2&amp;deg; display)</td><td>Elecrow</td><td>Elecrow 10.1 Inch Raspberry Pi 1920x1080p Resolution Display</td><td></td></tr><tr><td>14&quot; Business Class Laptop 5490</td><td>Dell</td><td>84 / rcrc961481-4860836</td><td></td></tr><tr><td>20&quot; x 50&quot; Absorbant Liners</td><td>Fisher Scientific</td><td>AL2050</td><td>works well to protect floor of arena, could use any type of liner</td></tr><tr><td>21.5&quot; monitor (1&amp;deg; display)</td><td>Acer</td><td>Acer R221Q bid 21.5-inch IPS Full HD Display</td><td></td></tr><tr><td>CCD Camera</td><td>Lumenera Corporation</td><td>Infiniyy3S-1UR</td><td>excellent for behavioral studies due to high fps rate (60 fps)</td></tr><tr><td>Enclosure (alminum frames and PVC panels)</td><td>80/20 Inc.</td><td>4x cat.#9010, 4x cat.#9005, 1x cat.#9000, 5x cat.#65-2616</td><td>excellent, used quick build tab to find PVC, joints, and frame</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>22-032-601</td><td></td></tr><tr><td>Excel Spreadsheet Software</td><td>Microsoft Office</td><td></td><td>user friendly and widespread knowledge of Microsoft Office software</td></tr><tr><td>Freearm</td><td>Amazon</td><td></td><td>used to mount camera to the table, could use any mountable extendable arm</td></tr><tr><td>ImagePro Premiere 3D</td><td>Media Cybernetics</td><td>version 9.3</td><td>good program, could use some updating with the automated tracking feature</td></tr><tr><td>Matlab software (Psychotoolbox 3)</td><td>MathWorks</td><td>Matlab R2018b 64-bit (9.5.0.944444)</td><td>excellent software to generate pattern stimuli of any conditions</td></tr><tr><td>SteamPix sorftware</td><td>Norpix</td><td>StreamPix 7 64-bit Single Camera</td><td>works well, a few problems with frame dropping but good customer service</td></tr><tr><td>WD My Book External Hard Drive</td><td>Western Digital</td><td>WDBBGB0080HBK hard drive 8 TB USB 3.0</td><td>necessary if using .avi files with no compression codec due to large size of files</td></tr><tr><td>Wide angle lens</td><td>Navitar</td><td>NMV-5M23</td><td>excellent and necessary to capture entire arena</td></tr></tbody></table>

using looming visual stimuli to evaluate mouse vision

Using Looming Visual Stimuli to Evaluate Mouse Vision

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina through the lateral geniculate nucleus to the visual cortex, the system is complex. Cellular and molecular studies have been conducted to elucidate the mechanisms underpinning visual processing and, by extension, disease mechanisms. These studies may contribute to the development of artificial visual systems. To validate the results of these studies, behavioral vision testing is necessary. Here, we show that the looming stimulation experiment is a reliable mouse vision test that requires a relatively simple setup. The looming experiment was conducted in a large enclosure with a shelter in one corner and a computer monitor located on the ceiling. A CCD camera positioned next to the computer monitor served to observe mouse behavior. A mouse was placed in the enclosure for 10 minutes and allowed to acclimate to and explore the surroundings. Then, the monitor projected a program-derived looming stimulus 10 times. The mouse responded to the stimuli either by freezing or by fleeing to the hiding place. The mouse&#39;s behavior before and after the looming stimuli was recorded, and the video was analyzed using motion tracking software. The velocity of the mouse movement significantly changed after the looming stimuli. In contrast, no reaction was observed in blind mice. Our results demonstrate that the simple looming experiment is a reliable test of mouse vision.

A mouse with healthy eyes was placed in the enclosure and allowed to acclimate for 10 min. The arena with the monitor on the ceiling was kept under mesopic light conditions (7 x 105 photons/μm2/s). During the acclimation period, the mouse explored the space and found the opaque dome as a refuge. When the mouse moved away from the refuge, video capturing started, followed by initiation of the visual stimulus. In response to the looming stimulus, most mice ran into the dome (flight response), which wa...

To examine mouse vision, we conducted a looming test. Mice were placed in a large square arena with a monitor on its ceiling. The looming visual stimulus consistently evoked freezing or flight reactions in the mice.

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina ...

using-looming-visual-stimuli-to-evaluate-mouse-vision

Research

JoVE Journal

Behavior

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

Source: Maruoka, H., et. al. Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice. J. Vis. Exp. (2022)     This video shows a two-photon microscopy procedure for imaging neuronal activity and microglial dynamics in an awake mouse during visual stimulation, highlighting calcium transients and cellular interactions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Source: Bosco, A., et al. In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma. J. Vis. Exp. (2015) This video demonstrates in vivo imaging of retinal microglia in a mouse model of glaucoma. A transgenic mouse with fluorescent protein-expressing retinal microglia is positioned under an ophthalmoscope. Infrared illumination is used to locate the optic nerve head (ONH), and the focal plane is...

In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Here, we present detailed protocols for monocular visual deprivation and ocular dominance plasticity analysis, which are important methods for studying the neural mechanisms of visual plasticity during the critical period and the effects of specific genes on visual development.

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

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

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.

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

Learn looming visual stimuli testing for mouse vision using behavioral assays. This robust method evaluates image forming pathways through freezing and flight responses without extensive training.

Using Looming Visual Stimuli to Evaluate Mouse Vision

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