Name: the optokinetic response as a quantitative measure of visual acuity in zebrafish
Uploaded: 2013-10-09T22:00:00.000+00:00
Duration: 4 min 56 s
Description: Watch this Scientific Journal Video about The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish at JoVE.com

Funding provided by the Western University of Health Sciences College of Optometry to DJC. Special thanks to Irisa Tam for graphic design.

All animal husbandry and experiments should be approved and conducted in accordance with guidelines set forth by the Institutional Animal Care and Use Committee of the respective institution or other legal requirements.
1. Zebrafish Care
<ol>
	<li>Maintain zebrafish under standard conditions at 28.5 °C on a 10 hr dark-14 hr light cycle16.</li>
	<li>Keep adult zebrafish at a tank density of about 3 fish/L.</li>
</ol>
2. Optokinetic response (OKR)
<ol>
	<li>Build the custom OKR recording device using a 14.5 cm diameter rotating drum, a stereo microscope with adjustable light intensity settings (300-8,000 lux) and a computer (Figure 1a).
	<ol>
		<li>Attach a camera to the microscope that will provide a live feed onto an adjacent monitor and allow for image capture and recording.</li>
		<li>Control the rotating drum using a microcontroller linked to a computer which will accommodate various speed and direction selections.</li>
	</ol>
	</li>
	<li>Anesthetize the fish in 0.016% tricaine for 2-3 min and then place the fish on a small platform with the eyes and gills suspended over the edge.</li>
	<li>Place a thin sponge/towel over the body of the fish and pin 2-3 pieces of foam, shaped to accommodate the fish, over the fish to keep it immobilized. Limiting the movement of the tail of the fish was found to be a crucial aspect for immobilization without causing injury to the fish, and was best achieved using a flat piece of foam. (Figure 1b)</li>
	<li>Position the fish within a cylindrical water-filled tank, that fits inside the rotating drum of the OKR recording device. Use magnets on the platform to position the fish upright, with the eyes approximately 7.3 cm from the edge of the drum. The fish should revive from the anesthesia within a few minutes - normal breathing will resume and random eye movements should be observed.</li>
	<li>Place a base grating of 0.07 cycles per degree (cpd) into the rotating drum and engage the computer controls to begin rotation and video capture (a speed of 12 rpm was used for this paper, but speeds of 8-16 rpm should give similar results).</li>
	<li>Once an initial OKR is elicited by the base grating, pause the rotation briefly, and replace the grating with a smaller grating (higher spatial frequency).</li>
	<li>Repeat this process until an OKR can no longer be elicited. Retest with the smallest grating that caused an OKR following a modified staircase approach and then repeat using the grating that failed to elicit a response to verify the loss of the OKR. This process may be repeated to verify a true response to the smallest detectable grating if the final grating changed relative to the first extinction event.</li>
	<li>Obtain monocular acuity measurements by placing a black plastic occluder over the stripes adjacent to the opposite eye. Repeat the procedure above (steps 2.6-2.7).</li>
	<li>Obtain the acuity of the opposite eye by repositioning the occluder and repeating the steps 2.6-2.7 again.</li>
	<li>Note the distance each eye is from the stripes during the testing procedure by using the reference measuring strip beneath the fish tank so that accurate acuity measures can be calculated in the following steps.</li>
</ol>
3. Calculating Visual Acuity
<ol>
	<li>Obtain the visual acuity by calculating the cpd: <img alt="Mathematical expression of 1 over 2 times arctan of h over 2a; equation for angle calculation." fo:content-width="0.7in" src="/files/ftp_upload/50832/50832eq1.jpg" width="70px" data-altupdatedat="1747911167000" data-alt="Equation 1"> where a is the distance from the center of the lens to the grating, and h is the length of one cycle of the smallest grating at which OKR was observed (Figure 1c).</li>
	<li>For combined visual acuity measurements, the averaged a-value from both the left and right eye is used.</li>
</ol>

<ol>
	<li>Brockerhoff, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+behavioral+screen+for+isolating+zebrafish+mutants+with+visual+system+defects.">A behavioral screen for isolating zebrafish mutants with visual system defects.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 10545-10549 (1995).</li><li>Neuhauss, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disorders+of+vision+revealed+by+a+behavioral+screen+of+400+essential+loci+in+zebrafish.">Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish.</a> J. Neurosci. 19, 8603-8615 (1999).</li><li>Schumann, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+objective+determination+of+visual+acuity+on+the+basis+of+the+optokinetic+nystagmus.">The objective determination of visual acuity on the basis of the optokinetic nystagmus.</a> Am. J. Optom. Arch. Am. Acad. Optom. 29, 575-583 (1952).</li><li>Gorman, J. J., Cogan, D. G., Gellis, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+apparatus+for+grading+the+visual+acuity+of+infants+on+the+basis+of+opticokinetic+nystagmus.">An apparatus for grading the visual acuity of infants on the basis of opticokinetic nystagmus.</a> Pediatrics. 19, 1088-1092 (1957).</li><li>Fulton, A. B., Manning, K. 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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+optokinetic+response+to+study+visual+function+of+zebrafish.">Using the optokinetic response to study visual function of zebrafish.</a> J. Vis. Exp. (36), e1742 (2010).</li><li>Mueller, K. P., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurements+of+the+optokinetic+response+in+adult+fish.">Quantitative measurements of the optokinetic response in adult fish.</a> J. Neurosci. Methods. 186, 29-34 (2010).</li><li>Li, L., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dominant+form+of+inherited+retinal+degeneration+caused+by+a+non-photoreceptor+cell-specific+mutation.">A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 11645-11650 (1997).</li><li>Ren, J. Q., McCarthy, W. R., Zhang, H., Adolph, A. R., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+visual+responses+of+wild-type+and+hypopigmented+zebrafish.">Behavioral visual responses of wild-type and hypopigmented zebrafish.</a> Vision Res. 42, 293-299 (2002).</li><li>Maaswinkel, H., Mason, B., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ENU-induced+late-onset+night+blindness+associated+with+rod+photoreceptor+cell+degeneration+in+zebrafish.">ENU-induced late-onset night blindness associated with rod photoreceptor cell degeneration in zebrafish.</a> Mech. Ageing Dev. 124, 1065-1071 (2003).</li><li>Stujenske, J. M., Dowling, J. E., Emran, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bugeye+mutant+zebrafish+exhibits+visual+deficits+that+arise+with+the+onset+of+an+enlarged+eye+phenotype.">The bugeye mutant zebrafish exhibits visual deficits that arise with the onset of an enlarged eye phenotype.</a> Invest. Ophthalmol. Vis. Sci. 52, 4200-4207 (2011).</li><li>Veth, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+zebrafish+lrp2+result+in+adult-onset+ocular+pathogenesis+that+models+myopia+and+other+risk+factors+for+glaucoma.">Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma.</a> PLoS Genet. 7, e1001310 (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK, A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Cagenello, R., Arditi, A., Halpern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binocular+enhancement+of+visual+acuity.">Binocular enhancement of visual acuity.</a> J. Opt. Soc. Am. A Opt. Image Sci. Vis. 10, 1841-1848 (1993).</li><li>Brockerhoff, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+optokinetic+response+of+zebrafish+larvae.">Measuring the optokinetic response of zebrafish larvae.</a> Nat. Protoc. 1, 2448-2451 (2006).</li><li>Hodel, C., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-based+analysis+of+the+optokinetic+response+in+zebrafish+larvae.">Computer-based analysis of the optokinetic response in zebrafish larvae.</a> CSH Protoc. 2008, pdb prot4961 (2008).</li><li>Huber-Reggi, S. P., Mueller, K. P., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+optokinetic+response+in+zebrafish+by+computer-based+eye+tracking.">Analysis of optokinetic response in zebrafish by computer-based eye tracking.</a> Methods Mol. Biol. 935, 139-160 (2013).</li><li>Tappeiner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+contrast+sensitivity+of+adult+zebrafish.">Visual acuity and contrast sensitivity of adult zebrafish.</a> Front. Zool. 9, 10 (2012).</li><li>Saszik, S., Bilotta, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+on+the+dark-adapted+spectral+sensitivity+function+of+the+adult+zebrafish.">The effects of temperature on the dark-adapted spectral sensitivity function of the adult zebrafish.</a> Vision Res. 39, 1051-1058 (1999).</li><li>Haug, M. F., Biehlmaier, O., Mueller, K. P., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+in+larval+zebrafish:+behavior+and+histology.">Visual acuity in larval zebrafish: behavior and histology.</a> Front. Zool. 7, 8 (2010).</li><li>Curcio, C. A., Sloan, K. R., Kalina, R. E., Hendrickson, A. 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The authors have nothing to disclose.

On occasion the fish may escape into the water tank. It is critical to immobilize the tail. Additionally the use of a thin sponge/towel over the fish&#39;s body was also found to be invaluable to keeping the fish suspended for prolonged periods of time. We were able to keep fish immobilized for more than 30 min using this procedure without harming the fish. This amount of time is more than sufficient to do the binocular and both monocular acuities.
The presentation of the grating can be either done as described here and elsewhere using a physically rotating drum or by presenting a digital display18-22. Both types have specific advantages and disadvantages. We chose to use the physical rotation both for cost reasons and technical specifications. Excluding the computer and imaging equipment, the OKR device described here can be built for around $150.
The OKR is an objective means of determining visual acuity in adult zebrafish. While several other visuomotor assays for vision have been used in previous experiments and even the electroretinogram has been successfully done in adult zebrafish eyes, though it has not been heavily utilized, the adult zebrafish OKR opens the door to many new experimental conditions22. This study has demonstrated the OKR&#39;s utility in measuring visual acuity changes over time, identifying visual acuity difference in eye disease models, and in determining monocular visual acuity.
Interestingly, adult zebrafish vision when compared to human vision (20/20) is approximately 20/1,000 (when 0.60 cpd is converted to -0.3 logMAR). Although this may seem poor, the zebrafish eyes are very well suited for its environment. Despite the visual task of looking through 6.5 cm of water, a few millimeters of polycarbonate, and another centimeter of air, the fish perform quite well! We found that the adult zebrafish acuity is about 70% of what would be predicted given their photoreceptor spacing.23 Contrast that with optimal human performance ~20/12, which is about 80% of the predicted 20/10, and OKR technique for measuring visual capability in adult zebrafish is impressive24,25.
The technique of measuring independent eye visual acuities would allow for studies in which one eye could be treated and the other used as a control. Additionally, fish with asymmetric eye development such as the lrp2/bugeye or fish that develop cataract could be monitored more accurately. It should be noted that care needs to be taken to accurately measure the distance from the eyes to the stripes and to occlude the full field of vision. This technique should open the door to more accurate and specific measures of zebrafish visual function.

Zebrafish (Danio rerio) are a good model to study visual physiology due to the similarity of their retina to other vertebrates, their short life cycle, and the availability of genetically altered mutants1,2. The optokinetic reflex/response/nystagmus (OKR) is a combination of smooth pursuit and rapid saccade eye movements. For more than 60 years clinicians have demonstrated that the OKR can be used for objectively measuring visual acuity in patients and is especially useful for determining infant visual capabilities3-5. The first recorded use of OKR in animals was using pigeons in the 1950s6. More recently, the OKR has become a valuable tool for assessing the visual function in larval zebrafish and is often used to screen for genetic mutants that have a visual impairment1,2. While the OKR has been widely used to determine visual function in larval zebrafish, it has only recently been demonstrated in adult zebrafish7-10. A recent paper by Tappeiner et al. uses a commercially available optomotor system traditionally used for mice, OptoMotry, to demonstrate that the visual acuity of the adult zebrafish remains quite constant, ~0.59 cycles per degree (cpd), even across various angular velocities7.
Although the OKR has proven to be quite useful in larval zebrafish studies, other methods for visual behavior have been used in zebrafish adults with varying degrees of success. The escape response assay demonstrated that albino and ruby zebrafish, both pigmentation mutants, have reduced visual responses in bright light conditions11,12. This same escape response assay has also successfully identified night blindness d mutants, albeit at 2 years of age13. However, the escape response assay is not without flaws. It is difficult to ascribe to an exact visual function and is only a gross approximation of visual changes - meaning it takes a large change before the change is identified.
Another method that has been developed for identifying adult zebrafish with visual impairments is the optomotor response (OMR)11. In this assay, fish are placed in a circular tank with an opaque column in the center. Black and white stripes rotate around the tank and the fish is tasked with swimming in the direction of the stripe motion. Like the escape response, the OMR focuses on the visuomotor capabilities of the adult zebrafish. It has however been used successfully to identify fish with visual impairments, such as the lrp2/bugeye mutants14. The lrp2/bugeye zebrafish exhibit buphthalmos, elevated intraocular pressure, diminished OMR, and progressive retinal degeneration14,15.
Many studies have used the visuomotor capabilities in adult zebrafish, which are often subjective and cannot be quantitatively analyzed. By using the OKR, one can study the visual acuity of adult fish more objectively. We have built our own OKR device, modeled after one initially used for larval studies1. In this study we demonstrate that the OKR can be used to calculate both monocular and binocular visual acuity in zebrafish.

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the optokinetic response as a quantitative measure of visual acuity in zebrafish

Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression.

OKR Device Functionality
The OKR device as explained above and shown in Figure 1 functions with minimal maintenance. As noted in step 2.3, immobilizing the tail is critical to keeping the fish restrained during the recording. Sufficiently immobilized, the fish can be kept in the OKR device for prolonged periods of time. Consequently, care should be taken during the initial immobilization steps, so that more data can be collected for each individual fish.
Visual acuity measurements
The binocular OKR is readily elicited in normal adult fish. By following the procedure above one can find visual acuities for zebrafish of many ages (Figure 2). The best binocular spatial acuity we recorded was 0.74 cpd. As noted in Figure 2, the binocular acuity generally increases with age (n=46; p = 0.002 ANOVA using SPSS v14), but levels off after about 12 months of age. When we compared the measured visual acuity in 5 month old wild type fish to age-matched lrp2/bugeye fish, we found that the lrp2/bugeye fish had significantly reduced acuity (p &lt; 0.001; wild type: 0.49±0.05 cpd; n=8; lrp2/bugeye: 0.35±0.06; n=10; Student's T-test using Excel).
By occluding the right eye the visual acuity for the left eye can be determined and vice versa. When compared to the binocular acuity, binocular summation is observed similarly to human vision17 (Figure 3). The binocular acuity was generally 5-10% better than either right (OD) or left (OS) acuities measured independently. For some individual fish the difference was a 25% improvement.
<img alt="Microscopy system with spatial grating, rotating drum, light control, and schematic for optical study." fo:content-width="6in" src="/files/ftp_upload/50832/50832fig1.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. OKR recording. A) The OKR device consists of a 14.5 cm diameter rotating drum and microscope with adjustable light intensity settings (300-8,000 lux). A camera provides a live feed on an adjacent monitor. The rotating drum has various speed settings in both clockwise and counterclockwise directions and interchangeable spatial frequency gratings are inserted and removed as needed. B) Fish are secured and placed in the center of the rotating drum. C) Cycles per degree (cpd) is calculated using cpd=<img alt="Optics formula 1/2 tan⁻¹(h/2a) related to lens focusing and light refraction." src="/files/ftp_upload/50832/50832eq2.jpg" data-altupdatedat="1747911167000" data-alt="Equation 2"> <a href="https://www.jove.com/files/ftp_upload/50832/50832fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Visual acuity development graph; cycles/degree vs. age in months; study of eyesight maturation." fo:content-width="6in" src="/files/ftp_upload/50832/50832fig2.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Visual acuity changes with age. The graph shows independent visual acuities measured 5-15 months in age. The visual acuity generally increases throughout the first year of development and then tails off a bit at 15 months of age (p = 0.002 ANOVA using SPSS v14). Each point may either represent 1 or several fish. Visual acuity is measured in cycles/degree (cpd). (n= 46 total; 8 at 5 months; 4 at 6 months; 7 at 8 months; 6 at 9 months; 11 at 10 months; 2 at 11 months; 2 at 12 months; and 6 at 15 months). <a href="https://www.jove.com/files/ftp_upload/50832/50832fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Binocular visual acuity summation effect bar chart; cycles/degree measurement comparison." fo:content-width="6in" src="/files/ftp_upload/50832/50832fig3.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Binocular visual acuity summation effect. The binocular (OU) visual acuity is generally higher compared to right (OD) or left (OS) eye acuities. In this particular group the OS acuities were significantly lower. (* p=0.006, N=8) <a href="https://www.jove.com/files/ftp_upload/50832/50832fig3highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish at JoVE.com

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

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20.4K Views.  Western University of Health Sciences. Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

Video: The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biology

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

A New Optokinetic Testing Method to Measure Rat Vision

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement associated with OKN is commonly used as a measure of visual function in rodents. To record OKN responses in normal and experimental rats, a simple and inexpensive apparatus has been developed. This setup uses two tablet screens to display the OKN visual stimulus consisting of high contrast black and white stripes generated using the OKN Stripes Visualization Web Application, a freely available software. The rat is placed inside a clear Plexiglass holder that limits&#160;movement so that the&#160;rat&#39;s head continuously faces the OKN display screen. The position of the rat holder can be changed to adjust the distance between the rat and the display screen. A micro-camera positioned above the rat holder is used to record the rat&#39;s visual activities. These recordings can be used for quantitative assessments. Based on the presence or absence of clear head-tracking, the OKN responses at different spatial frequencies can be determined. The collected data demonstrates a novel technique for reliable measurement of visual acuity in normal and retinal degenerate rats.

The Optokinetic Nystagmus (OKN) behavioral testing method is used for the assessment of visual acuity in rodents. Here a simple method is demonstrated that can be easily set up in research laboratories for a reliable assessment of visual function in both normal and experimental rats.

Optokinetic nystagmus (OKN) is a reflexive eye movement initiated by the motion of visual stimuli in the field of vision. The head-tracking movement ...

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 ...

Photomotor Response Assay: A Method to Measure the Behavioral Response of Larval Zebrafish to a Sudden Change in Lighting Condition

Source: Steele, W. B., et al. <a href="https://www.jove.com/v/57938/" target="_blank">Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine</a>.&#160;J. Vis. Exp.&#160;(2018).
This video describes the photomotor response assay of zebrafish and fathead minnow larvae using an automated tracking software. The protocol measures the behavioral response of the larval fish following a sudden change in lighting condition.

Test odpowiedzi fotomotorycznej: metoda pomiaru reakcji behawioralnej larw danio pręgowanego na nagłą zmianę warunków oświetleniowych

Source: Steele, W. B., et al. Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant ...

JoVE Encyclopedia of Experiments

Danio rerio (zebrafish)

Larva

Optokinetic Response Assay: A Method to Measure Visual Acuity in Zebrafish

Source: Cameron, D. J., et al. <a href="https://www.jove.com/v/50832/" target="_blank">The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish</a>.&#160;J. Vis. Exp.&#160;(2013)
This video describes the optokinetic response assay in zebrafish. The video measures the reflexive eye movements based on the visual moving stimulus and determines the visual acuity in zebrafish.

Test odpowiedzi optokinetycznej: metoda pomiaru ostrości wzroku u danio pręgowanego

Source: Cameron, D. J., et al. The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish.&#160;J. Vis. Exp.&#160;(2013)
This video ...

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

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VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Using the optokinetic response to study visual function of zebrafish

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

A New Optokinetic Testing Method to Measure Rat Vision

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

Test odpowiedzi fotomotorycznej: metoda pomiaru reakcji behawioralnej larw danio pręgowanego na nagłą zmianę warunków oświetleniowych

Test odpowiedzi optokinetycznej: metoda pomiaru ostrości wzroku u danio pręgowanego