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 t....

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; ....

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