Using Looming Visual Stimuli to Evaluate Mouse Vision

Podsumowanie

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.

Streszczenie

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'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.

Wprowadzenie

The visual system starts at the retina, where visual signals are captured by photoreceptors, channeled to bipolar cells (2^nd-order neurons), and finally passed to ganglion cells (3^rd-order neurons). Retinal 2^nd- and 3^rd-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 processing^1,2,3,4,5,6. Recently, more than 15 types of bipolar cells^7,8,9,10,11 and ganglion cells^{12,13,14,15,16} 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 genes^8,10,17,18, 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 conditions^21,22. 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 mouse^28,29, zebrafish³⁰, locust^31,32, and human^33,34,35. 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 circuit^36,37,38. 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.

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Protokół

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

1. 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.
2. Set up a camera with a wide-angle lens for capturing the mouse’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.
3. 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.
4. 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° and expand to 50° 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.
5. 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.

2. Mouse acclimation

1. 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.
2. Allow the mouse to acclimate anywhere from 7 to 15 min^29,39. We allowed 10 min of acclimation prior to stimulus onset. Furthermore, 10 min acclimation one day prior to the experiment may ease the mice.

3. Looming visual stimuli projection

1. 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.
2. 10 seconds prior to the stimulation, start the video capturing.
3. 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.
   1. 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.
4. 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.

4. Video analysis

1. 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.
2. Use analysis software to track the animal’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).
3. 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).
4. 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

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Wyniki

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), whi...

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Dyskusje

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)...

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Materiały

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