Name: using looming visual stimuli to evaluate mouse vision
Uploaded: 2019-06-13T14:30:00
Description: Watch this Scientific Journal Video about Using Looming Visual Stimuli to Evaluate Mouse Vision at JoVE.com

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
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	<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 ...

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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.

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			<td height="21" style="height:21px;width:299px;">10.1&#34; monitor (2&#176; display)</td>
			<td style="width:148px;">Elecrow</td>
			<td style="width:903px;">Elecrow 10.1 Inch Raspberry Pi 1920x1080p Resolution Display</td>
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			<td height="21" style="height:21px;">14&#34; Business Class Laptop 5490</td>
			<td>Dell</td>
			<td>84 / rcrc961481-4860836</td>
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			<td height="21" style="height:21px;">20&#34; x 50&#34; 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>
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			<td height="21" style="height:21px;">21.5&#34; monitor (1&#176; display)</td>
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			<td>Lumenera Corporation</td>
			<td>Infiniyy3S-1UR</td>
			<td>excellent for behavioral studies due to high fps rate (60 fps)</td>
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			<td>80/20 Inc.</td>
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			<td>excellent, used quick build tab to find PVC, joints, and frame</td>
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			<td height="21" style="height:21px;">Ethanol</td>
			<td>Fisher Scientific</td>
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			<td>Microsoft Office</td>
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			<td>user friendly and widespread knowledge of Microsoft Office software</td>
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			<td>Amazon</td>
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			<td>MathWorks</td>
			<td>Matlab R2018b 64-bit (9.5.0.944444)</td>
			<td>excellent software to generate pattern stimuli of any conditions</td>
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			<td height="21" style="height:21px;">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>
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			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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), whi...

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 ...

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