A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Summary

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.

Abstract

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.

Introduction

Xenopus laevis have been used as a model organism to study eye formation. The eyes develop quickly, growing to maturity in less than a week for testing genes or pathways that have an effect on visual development and function. To test visual function, the optokinetic and optomotor response has been used in zebrafish and Xenopus tadpoles, respectively^1,2 . Because the eyes of Xenopus tadpoles are relatively smaller than zebrafish, this assay requires the use of specialized equipment and trained personnel to detect the subtle tail-flip and eye-movement behavior in Xenopus. A more robust behavior in Xenopus is the preference for swimming in a tank with a white background, which is described herein³. When placing a tadpole in a half black/half-white tank, the pre-metamorphic tadpole quickly swims to the white side of the tank. We previously used this assay to determine if pluripotent cell-derived eyes were functional⁴. Here, we report a detailed version of this assay, which can be used to test the visual function of premetamorphic Xenopus tadpoles.

This assay is simpler than the optomotor response assay, since it only requires a mounted digital camcorder, digital camera software and standard equipment found in most Xenopus laboratories. In addition, the recorded response requires no special training to tally results. Our representative results show that the same group of tadpoles, having undergone double retinal axotomy, swim randomly around the tank. We have also included the behavior assay results from a representative tadpole, showing how one eye can be tested for visual response. A worksheet has been included so that numbers acquired during the assay can be inserted and analyzed. This worksheet can be used to determine whether the tadpoles tested have a visual response.

Protocol

Animal Care

The Xenopus laevis tadpoles used in this study were grown, raised and treated according to procedures approved by Upstate Medical University IACUC and the Guide for the Care and Use of Laboratory Animals.

1. Pre-behavior Setup: Tadpoles

1. Obtain fertilized Xenopus embryos from a commercially available source or in vitro fertilize oocytes, as previously described⁶.
2. Place blastula to neural plate stage embryos at 18 ˚C in 60 mm Petri dishes containing 0.1X MMR [10 X Marc's Modified Ringer's (MMR) solution stock (10 mM MgCl₂; 20 mM KCl; 20 mM CaCl₂ ; 50 mM HEPES; 1 M NaCl; adjusted to pH 7.5, autoclaved and stored at RT)] with 50 µg/ml gentamicin antibiotic. Change 0.1X MMR each day and remove any dead embryos.
3. Once the embryos reach Nieuwkoop and Faber stage ~27 - 30⁷, switch the embryos into 0.1X MMR without antibiotic to allow gut bacterial growth.
4. Grow the tadpoles until they reach the feeding stage (stage 45) and move them into 100 mm Petri dishes filled with 0.1X MMR. Feed them nettle powder supernatant every other day for a week, changing the 0.1X MMR on the intervening day.
5. After one week in the 100 mm Petri dish, move animals to half gallon tanks filled with frog water [0.5 g/L instant ocean and 2 mM Sodium phosphate dibasic (Na₂HPO₄: mw 141.96) pH 6.8]. Feed them as in step 2.4, but change water only 2-3 times per week when water is cloudy. Maintain the animals in a 12 hr light/12 dark light cycle with the lights turning on at 6 am.
6. Place the half-gallon tank with the test subjects on a white surface like a lab bench underpad, at least 12 hr O/N, prior to testing.
7. Test tadpoles at stages 45 to 50 as described in step 3 below.

2. Pre-behavior Setup: Equipment

1. Set aside an area of the lab for the behavior assay. It should be in a quiet, low-traffic area with standard fluorescent lighting.
2. Prepare the testing tanks for the animals.
   The nested, half-gallon testing tanks are composed of two parts: an inner tank that will hold the water and tadpoles; and an outer tank with the visual stimulus.
   1. For the inner tank, make a small mark on the outside corners with a sharpie, 5 cm from the bottom; this is the water line (Figure 1A, green dotted line).
   2. Also, for the inner tank, fill the divets in the tank (at the corners and the center) with an inert compound, like Sylgard elastomer. NOTE: The tadpoles will linger in these areas if they are not filled in.
   3. For the outer tank, cover exactly one half of the outside of one tank with black electrical tape and the other half with the white tissue paper Figure 1B.
3. Place the tanks on the inoculating turntable.
4. Setup the webcam/tripod so that it is above the testing tanks. Adjust the camera to allow visualization of the testing area, as shown in Figure 1C.
5. Connect the webcam to a computer with QuickTime Player software installed⁵. Turn on the camera. In QuickTime Player, under File, select ‘New Movie Recording.’ The behavior assay setup will be visible on the computer right away.
6. Drape a lightweight cotton cloth over the entire setup to reduce external cues that could affect tadpole behavior, as well as, reduce reflected light on the surface of the water.
7. Ensure that the luminance under the cloth measures between 35-50 cd/m².

3. Behavior Assay

1. Fill the inner testing tank to the 5 cm water mark made in step 1.2.1 with frog water.
2. Using a small net, gently move the animal into the inner testing tank.
3. Open QuickTime software on computer and, using the outer tank as a guide, make sure that the camera is able to visualize and record the testing area.
4. Write the animal name, date and time on a piece of paper and record with the camera.
5. Place the test tank into the outer tank with the black side of the tank to the right Figure 2.
6. Start recording the movie immediately after arranging the tank and set timer for 2 min.
7. When the timer beeps, remove the inner test tank, rotate the outer tank 180° and place the inner test tank inside the outer tank. Start the timer. Note: the black side should now be on the other side.
8. Repeat step 3.7, eight more times for a total of ten trials. Use Figure 2 as a guide, checking off each trial.
9. Repeat the behavior assay on two separate days.

4. Retinal Axotomy

1. Place animal in 0.02% tricaine until they are unresponsive to a tail pinch with a #3 forceps.
2. Melt 1% agarose in 0.1X MMR and then add to a 60 mm Petri dish. Once cooled, make a small rectangular divot in the agarose and add the tadpole to the divot with a little 0.02% tricaine, so that the animal is partially submerged in the liquid.
3. Pierce the skin at a 45 degree angle behind the dorsal region of the eye with a 25 G needle, while bracing the animal against forceps on the opposite side.
4. Taking care not to snip the vein, which lies next to the optic nerve, carefully reach into the hole with #5 forceps, snip the optic nerve and flip it out of the way. If there is severe bleeding due to accidently snipping the artery, then quickly place the animal in 2% tricaine to euthanize. CAUTION: 2% tricaine solution can cause numbness in humans. This solution should be handled with gloves.
5. To recover the animal after surgery, place animal in 100 mm Petri dish with 0.7X MMR and 50 µg/ml gentamicin for 20 min. Next, transfer tadpole to recovery tank with frog water. Let animal rest O/N.
6. The next morning, test the visual function as described above (steps 3.1-3.9).

5. Analyze Results

1. After the trials are finished, measure the amount of time the tadpole stays on the black side of the tank.
   1. Manually view the videos using software that displays the time and allows frequent pausing. Look at the video display time. Write down the start of the two-min trial, which is when the inner tank successfully nests inside the outer tank and is squared in view of the camera.
   2. Use the position of the tadpole’s eyes to define which side of the tank the animals are swimming on. Define crossing over from one side of the tank to the other only when both eyes have crossed the black/white line.
2. Write down the beginning and end of each interval (in sec) that the animal spends on the black side. Pause and rewind the video to ensure accuracy. Total the sec within each trial.
3. Input these numbers into the attached Behavior Worksheet. The worksheet will calculate the ratio of time that the animal spent on the white side by subtracting the time spent on the black side of the tank from the total 120 sec trial and divide by the total time in sec [(120 – total seconds spent on the black side)/120 sec]. The worksheet averages this ratio over all 10 trials.
4. Use a student’s T-test, paired, two-tailed distribution to determine if the trials between days are significant (P ≤ 0.05).

Results

Previous reports have shown that premetamorphic Xenopus tadpoles prefer to swim on the white side of a black/white tank and called this assay, the Background Color Preference Assay³ We have changed this assay in order to test the visual function of either eye of tadpoles in less than a week. In this way, their eyes can be collected for histological examination.

We show here how the response is due to visual cues. In Movie 1, the tadpoles in the left and rig...

Discussion

We report here a simple vision-guided behavioral assay that can be easily performed in under a week by minimally trained lab personnel. While other assays require specialized equipment and expertise in animal behavior, this assay allows a quick test to determine visual function. Another behavioral assay, the Visual Avoidance assay, has been developed to determine how the tectum contributes to visual perception in Xenopus¹⁰. This assay measures spatial tuning and contrast sensitivity in response to a m...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/2 Gallon Flex-Tank with Cover	eNasco	SB19271M	Size: 5-3/8" x 7" x 3-3/4"
Black electrical tape
White tissue paper
Large inoculating turntable	VWR	50809-022	Size: Dia 114.3 x  H 76.2 mm (4 1/2  x  3")
Durasorb underpad	VWR	82004-836	Size: 43.2 x 60.1 cm (17 x 24")
Kimwipe	Krackeler Scientific, Inc.	1945-34155-CS
Standard tripod		Various
iSight camera or webcam	Apple	M8817LL/A	Good for larger tadpoles but small ones are difficult to see
Portable computer	Apple/PC	Various	We used a 13" MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5
MiniDV handycam camcorder	SONY	DCR-HC42	Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400
Handycam station	SONY	DCRA-C121	This can be used for connecting firewire to camera
QuickTime Player software	Quicktime	Version 10.1
26 G Needle (5/8" length)	VWR	BD305115
Dumont #5 Forceps	Fine Science Tools	11295-10
Disposables
Gentamicin sulfate [50 mg/ml]	Fisher Scientific	17-528Z	Stored at RT
Sylgard 184 silicone elastomer	Fisher Scientific	NC9644388
Instant ocean	Doctors Foster and Smith	CD-116528	Stock solution = 100 g/L stored at RT
Sodium phosphate dibasic	Sigma Aldrich	S0876	Stock solution = 0.4 M stored at RT
In vitro fertilized embryos	eNasco	LM00490MX	100 embryos/unit