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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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). Taken together, we demonstrate that looming experiments are a useful and consistent vision test for mice.
We set the speed of the looming stimuli at 192 degree/s. Franceschi et al.29 examined the looming responses at varying speeds, 5 to 84 degree/s and observed freeze responses preferentially at lower speed levels. Yilmaz and Meister28 observed flight responses at 35 to 350 degree/s; however, flight latency was longer at higher speeds. Therefore, to evoke consistent flight responses, looming should be at a speed of 50 degree/s or above. Looming stimuli can be generated easily even with standard presentation software. However, such software cannot create higher speeds of looming stimuli. We instead used MatLab and PsychToolbox3 to create the visual stimuli at 192 degree/s.
We acclimated the mice for 10-15 min before the looming stimuli, which is the acclimation time previous researchers described 28,29,39. We furthermore tested whether acclimation the day before changed the looming behavior. We placed the mice in the enclosure for 10 min without looming stimuli the day before the looming stimuli. This acclimation significantly shortened the flight latency (p &#60; 0.01, n = 7 mice, data not shown). Although 10 min of acclimation on the day of looming consistently caused flight responses, 1 day prior acclimation decreased the latency of flight responses.
There are some limitations for using looming stimuli as a vision test. First, it is hard to test one eye at a time. Unless suturing the one eye, both eyes are tested together. Second, the mechanisms of the behavioral looming response have not been fully established. In the retina, Yilmaz and Meister 28 suggested that ventral OFF-DSGCs (direction-selective ganglion cells), but not ON-DSGCs, convey the looming signals to cause responses. This conclusion arose from their results that mice responded to dark looming stimuli, but not to white looming. In the brain, Wei et al.36 and Shang et al.37 demonstrated that the pathways from the superior colliculus through the amygdala and the periaqueductal gray are responsible for the looming. However, more studies should be conducted to confirm these investigations.
Even though some limitations exist with respect to the looming experiment, the looming visual stimulus generates consistent and robust fear response in mice and should be a useful test of mouse vision which requires minimal training of the experimenter.

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

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/&#956;m2/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 was observed in 30 out of 31 mice (97%). Some of the mice exhibited freeze responses before they fled (19/31 mice, 61%). The looming stimulus reduced the light condition 1 log (6 x 105 photons/&#956;m2/s).
Captured video clips were analyzed using either a commercial analytics software with a manual tracking function (Image Pro Plus) or FIJI (NIH). Using the tracking feature, the mouse&#8217;s position was identified in each frame of the video (60 frames/s) before, during, and after the looming stimuli. We analyzed the velocity changes over time as well as the distance to the shelter (Figure 3). When flight occurred, the velocity abruptly increased and the distance to the shelter reduced accordingly. In contrast, the velocity was near 0 mm/s when mice froze. The latency from the onset of the looming stimuli to flight ranged from 0.1 to 6.0 seconds (average 2.2 s, 30 mice). The range of maximum velocity for flight response was 500-3000 mm/s (30 mice).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59766/59766fig1.jpg" /> 
Figure 1: Experimental system. (A) Schematic of the looming stimuli enclosure. A computer monitor (21&#34;) covers the ceiling. There is an opaque dome in one corner of the enclosure in which a mouse may take refuge. A video monitor with a wide-angle lens captures the mouse behavior. (B) Overall view of our entire setup. The secondary monitor duplicates the image showing on the stimulus display. (C) Diagram of the looming stimulus. The looming stimulus begins at 2&#176; (1.15cm) and holds at this size for 250 ms. It then expands to 50&#176; (30.8cm) over the course of 250 ms and remains 50&#176; for an additional 500 ms. This 1s sequence then repeats 9 more times prior to terminating. (D) Diagram of stimulus calculations. The height of the cage dictates the necessary start and end size (in centimeters) of the stimulus in order to produce a stimulus that expands from 2&#176; to 50&#176; when directly above the mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59766/59766fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59766/59766fig2.jpg" /> 
Figure 2: Analysis calculations. Calculations to correct skew from wide-angle lens. Due to the placement of the camera, the floor of the arena appears as a trapezoid instead of a rectangle (left). Therefore, the X and Y coordinates of the mouse must be corrected to accurately analyze mouse position (mid). Using the geometry of congruent triangles, it is possible to find how much the x-coordinate must shift in order to correctly represent the mouse&#8217;s movement in the 3-dimensional space (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59766/59766fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59766/59766fig3.jpg" /> 
Figure 3: Representative responses to looming stimuli. (A) An example of a mouse&#8217;s movement tracked within the arena. A red circle shows the dome where the mouse fled to and stayed until the looming disappeared. 1 = mouse position starting point when video capturing started. 2 = movement prior to stimulus onset when the mouse explored the arena. 3 = looming stimulus started. The mouse dashed to the dome (shown by a red dashed line). 4 = the mouse stayed in the dome until and after termination of the stimulus. (B) Velocity changes as a function of time for this mouse. The dotted line indicates when the looming stimulation began. Stimulus duration is indicated by the yellow background. The full 10 second cycle is not shown here since the mice remained stationary in the refuge for the entire stimulus duration. (C) Distance from the dome over time for the same mouse in (A) and (B). (D) and (E) The velocity and distance to the dome for a mouse that exhibited the freeze reaction (freezing duration shown by the red double sided arrow) prior to flight. The velocity was reduced compared to the control (before looming). The distance to the dome did not change during this period. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59766/59766fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Using Looming Visual Stimuli to Evaluate Mouse Vision at JoVE.com

Using Looming Visual Stimuli to Evaluate Mouse Vision

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

Nanyang Technological University

Research

JoVE Journal

Behavior

11.1K Views.  Wayne State University School of Medicine. 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.

Video: Using Looming Visual Stimuli to Evaluate Mouse Vision

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

Neuroscience

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

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.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

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

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

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.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicine

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

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

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.

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a ...

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

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the cortex to the contralateral eye to a stimulus is much stronger than the response of the ipsilateral eye in the binocular segment of the mouse primary visual cortex (V1). During the mammalian critical period, suturing the contralateral eye will result in a rapid loss of responsiveness of V1 cells to contralateral eye stimulation. With the continuing development of transgenic technologies, more and more studies are using transgenic mice as experimental models to examine the effects of specific genes on ocular dominance (OD) plasticity. In this study, we introduce detailed protocols for monocular visual deprivation and calculate the change in OD plasticity in mouse V1. After monocular deprivation (MD) for 4 days during the critical period, the orientation tuning curves of each neuron are measured, and the tuning curves of layer four neurons in V1 are compared between stimulation of the ipsilateral and contralateral eyes. The contralateral bias index (CBI) can be calculated using each cell&#39;s ocular OD score to indicate the degree of OD plasticity. This experimental technique is important for studying the neural mechanisms of OD plasticity during the critical period and for surveying the roles of specific genes in neural development. The major limitation is that the acute study cannot investigate the change in neural plasticity of the same mouse at a different time.

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 is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

Developmental Biology

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

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

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

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

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

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.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

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

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 

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 ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

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

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

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.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Using Looming Visual Stimuli to Evaluate Mouse Vision

Summary

Explore More Videos

Chapters in this video

VisualEyes: A Modular Software System for Oculomotor Experimentation

Video-oculography in Mice

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

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

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

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

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

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

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