A Novel Method of Drug Administration to Multiple Zebrafish (Danio rerio) and the Quantification of Withdrawal

Summary

A novel method for reducing variability when exposing fish to drugs is explained. Fish exposed to various patterns of ethanol exposure were found to have altered anxiety levels during withdrawal in a light/dark scototaxic assay.

Abstract

Anxiety testing in zebrafish is often studied in combination with the application of pharmacological substances. In these studies, fish are routinely netted and transported between home aquaria and dosing tanks. In order to enhance the ease of compound administration, a novel method for transferring fish between tanks for drug administration was developed. Inserts that are designed for spawning were used to transfer groups of fish into the drug solution, allowing accurate dosing of all fish in the group. This increases the precision and efficiency of dosing, which becomes very important in long schedules of repeated drug administration. We implemented this procedure for use in a study examining the behavior of zebrafish in the light/dark test after administering ethanol with differing 21 day schedules. In fish exposed to daily-moderate amounts of alcohol there was a significant difference in location preference after 2 days of withdrawal when compared to the control group. However, a significant difference in location preference in a group exposed to weekly-binge administration was not observed.

This protocol can be generalized for use with all types of compounds that are water-soluble and may be used in any situation when the behavior of fish during or after long schedules of drug administration is being examined. The light/dark test is also a valuable method of assessing withdrawal-induced changes in anxiety.

Introduction

The zebrafish (Danio rerio) is a small teleost species originating in India that is a useful model organism for behavioral¹ and medical research^2,3. Zebrafish are also commonly used in the testing of various pharmacological substances in order to characterize their impact on behavior. Various dosages and schedules of drug administration have been used to investigate the behavior of the zebrafish after the administration of compounds such as stimulants⁴, anxiolytics⁵ and ethanol^6-8.

Our lab has investigated the effects of differing schedules of ethanol administration on zebrafis....

Protocol

All procedures and behavioral testing were approved by MacEwan University’s Animal Research Ethics Board under protocol number 06-11-12, which is in compliance with the Canadian Council for Animal Care’s guidelines for the care and use of experimental animals.

1. Prepare Dosing Tanks, Solutions, and Administration Schedule

1. Prepare an administration schedule such that the animals are dosed in the same environment and during the same time of day to avoid any confounds of time or biasing to visual stimuli.
2. Obtain as many identical, 1.5 L, clear polypropylene spawning tanks as necessary for the number of group ....

Results

To maintain accuracy and control in pharmacological studies with zebrafish it is important to time the duration of ethanol administration consistently and accurately as described above. Our procedure can increase the ease and throughput of the dosing procedure. The administration of ethanol on either a weekly-binge or daily-moderate schedule resulted in altered anxiety levels, measured with the light/dark test, compared to controls. When tested two days after the last dosing, zebrafish in the control group (who received .......

Discussion

Previous studies involving drug administration in zebrafish have simply relied on netting fish to transport them from their home tank into the drug solution^12,16. Netting is not always consistent and often takes longer than expected due to the escape response of the zebrafish, which has significant individual variability. Traditional transfer methods, while useful, can be improved upon by decreasing the amount of total time the fish spend outside of water, as well as decreasing the amount of variability in tra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Three shelf benchtop housing system	Aquatic Habitats	N/A
1.5 L Spawning tank w/400 μm baffle	Aquatic Habitats	N/A
Pure Grain Ethanol	Luxco, INC	N/A
Ethovision XT Motion tracking software	Noldus Information Technology
Pipette	Eppendorf Canada
Light/Dark Arena	Custom		Construct as per procedure description. 9.5 cm wide, 9.5 cm deep, 55 cm long.