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

Summary

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.

Abstract

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.

Protocol

This protocol outlines the steps to perform the Visual-Motor Response of zebrafish larvae to light increments and decrements in your own lab. Zebrafish are a great model system for behavioral studies. They are easy to maintain, they have large clutch sizes, and they develop quickly. For example, the eyes of zebrafish larvae are responsive to light by day 3 of development and at which time they exhibit a startle response.

Part 1: Plating individual fish into a 96 well plate

1. Grow WT larvae under a dark/light cycle at 28°C until at least 4 days post fer....

Discussion

The experimental procedures we show in the film are all representative of WT fish. However, these experiments can be done analogously on mutant fish as well (see representative result section). We suggest to plate WT fish and mutant fish on the same plate in a checker board outline for optimal control purposes.

When using mutant fish on the same plate as WT fish make sure that you write down what type of fish was plated in each well. Here is a suggestion on how to keep track of the fish plate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microplate devices	Tool	Whatman, GE Healthcare	7701-1651
Transfer pipetes	Tool	VWR international	202205
Fish water	Reagent			refer to reference #4
Recording chambers (Zebrabox)	Tool	Viewpoint Lifesciences
Videotrack Software	Tool	Viewpoint Lifesciences