Name: the optokinetic response as a quantitative measure of visual acuity in zebrafish
Uploaded: 2013-10-09T22:00:00
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 &#176;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="Equation 1" fo:content-width="0.7in" src="/files/ftp_upload/50832/50832eq1.jpg" width="70px" /> 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. A., Dobson, V. <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+method+for+efficient+screening+of+visual+acuity+in+young+infants.+II.+Clinical+application.">A behavioral method for efficient screening of visual acuity in young infants. II. Clinical application.</a> Invest. Ophthalmol. Vis. Sci. 17, 1151-1157 (1978).</li><li>Huizinga, E., Van Der Meulen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+rotatory+and+optokinetic+reactions+in+the+pigeon.">Vestibular rotatory and optokinetic reactions in the pigeon.</a> Ann. Otol. Rhinol. Laryngol. 60, 927-947 (1951).</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>Mueller, K. P., Schnaedelbach, O. D., Russig, H. D., 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=VisioTracker,+an+innovative+automated+approach+to+oculomotor+analysis.">VisioTracker, an innovative automated approach to oculomotor analysis.</a> J. Vis. Exp. (56), e3556 (2011).</li><li>Zou, S. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+photoreceptor+topography.">Human photoreceptor topography.</a> J. Comp. Neurol. 292, 497-523 (1990).</li><li>Kalloniatis, M., Luu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Visual Acuity. , (1995).</li></ol>

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 &#60; 0.001; wild type: 0.49&#177;0.05 cpd; n=8; lrp2/bugeye: 0.35&#177;0.06; n=10; Student&#39;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="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50832/50832fig1.jpg" width="500px" /> 
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="Equation 2" src="/files/ftp_upload/50832/50832eq2.jpg" /> <a href="https://www.jove.com/files/ftp_upload/50832/50832fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/50832/50832fig2.jpg" width="500px" /> 
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="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50832/50832fig3.jpg" width="500px" /> 
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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Neuroscience

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

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Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

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 ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

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 ...

Assessing Vision via Shape Recognition in Random Dot Kinematograms

Motion-Acuity Test for Visual Field Acuity Measurement with Motion-Defined Shapes

The standard visual acuity measurements rely on stationary stimuli, either letters (Snellen charts), vertical lines (vernier acuity) or grating charts, processed by those regions of the visual system most sensitive to the stationary stimulation, receiving visual input from the central part of the visual field. Here, an acuity measurement is proposed based on discrimination of simple shapes, that are defined by motion of the dots in the random dot kinematograms (RDK) processed by visual regions sensitive to motion stimulation and receiving input also from the peripheral visual field. In the motion-acuity test, participants are asked to distinguish between a circle and an ellipse, with matching surfaces, built from RDKs, and separated from the background RDK either by coherence, direction, or velocity of dots. The acuity measurement is based on ellipse detection, which with every correct response becomes more circular until reaching the acuity threshold. The motion-acuity test can be presented in negative contrast (black dots on white background) or in positive contrast (white dots on black background). The motion defined shapes are located centrally within 8 visual degrees and are surrounded by RDK background. To test the influence of visual peripheries on centrally measured acuity, a mechanical narrowing of the visual field to 10 degrees is proposed, using opaque goggles with centrally located holes. This easy and replicable narrowing system is suitable for MRI protocols, allowing further investigations of the functions of the peripheral visual input. Here, a simple measurement of shape and motion perception simultaneously is proposed. This straightforward test assesses vision impairments depending on the central and peripheral visual field inputs. The proposed&#160;motion-acuity test advances the capability of standard tests to reveal spare or even strengthened vision functions in patients with injured visual system, that until now remained undetected.

Author Spotlight: Assessment of Visual Acuity in Central Vision Loss Through Motion-Based Peripheral Vision Testing

A novel motion-based acuity test that allows the assessment of central and peripheral visual processing in low-vision and healthy individuals, along with goggles limiting peripheral vision compatible with MRI protocols, are described here. This method offers a comprehensive vision assessment for functional impairments and dysfunctions of the visual system.

The standard visual acuity measurements rely on stationary stimuli, either letters (Snellen charts), vertical lines (vernier acuity) or grating charts, ...