Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

Summary

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

Abstract

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Introduction

The number of patients suffering from mood disorders, including anxiety-disorder and depression, is increasing. Although the development of these disorders involves both genetic and environmental factors, the detailed pathogenic mechanisms are not yet fully understood¹. Therefore, research needs to focus on uncovering the underlying mechanisms of mood disorders, as well as the development of effective drugs that have fewer side effects.

Behavioral tests for laboratory animals are useful for investigating brain higher function, including emotion. As animal behavior is highly variable, experimental outcomes often diff....

Protocol

All experimental procedures described here have been approved by the Institutional Animal Care and Use Committee of Meiji University (IACUC 14-0002(1)). Animals used in the representative results were prepared as follows: Male Sprague-Dawley rats were purchased from a laboratory animal company and housed in groups of 4 animals per standard rat cage (W200 × D410 × H250 mm) for at least one week before the start of the behavioral experiment. The animal housing room was maintained at standard ambient conditions for light (12:12 light/dark schedule with lights on at 10:00 h), room temperature (25.0 ± 0.5 °C), humidity (55 ± 10%), and ventilat....

Results

In the previous study, we compared the combined effects of open arm ledges and transparency of closed arm walls on the exploratory behaviors on the maze¹². Rats significantly spent much less time on open arms without ledges than on open arms with ledges (F[1, 60] = 13.49, P = 0.0005) (Figure 2A). Similar results were also found for the number of entries into open arms (F[1, 60] = 11.17, P < 0.0014)

Discussion

As the number of patients suffering from psychiatric disorders, including anxiety-disorder, increases, it is important to develop cutting-edge studies to improve understanding of the basic mechanism of anxiety. To achieve this, it is also important to optimize the experimental procedures. Therefore, to improve the application of the elevated plus maze test, we have demonstrated the effect of arm features on experimental outcomes using rats.

The elevated plus maze was originally established to .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Customized container (W1500 × D1500 × H2000 mm)	NA	NA	Handmade by Mr. Akihiro Hirano
Lighting	NA	NA	Handmade by Mr. Akihiro Hirano
Elevated plus maze apparatus	NA	NA	Handmade by Mr. Akihiro Hirano
Digital video camera	Watec	WAT-250D2	Any suitable video camera can be attached to a hard disk recorder for recording tracking profiles.
Hard disk recorder	TOSHIBA	RD-R200	Any suitable hard disk recorder can be attached to the digital video camera.
Sprague-Dawley rats	Charles River Laboratories Japan, Inc.	NA	Any suitable rats can be used for your study
Standard rat cage	Toyoriko	TP-105	Any suitable rat cage be used for your study
Instrument to measure light intensity	AS ONE	1-5818-02	Any suitable instrument to measure light intensity
EthoVision XT version 8.0	Noldus	NA	Any suitable software to measure animal behavior
Diazepam	Taiyo Pharmaceutical Industry	1124402A2061	Any suitable drug can be used for your study
Tween 80	Wako Pure Chemical Industries	9005-65-6	Any suitable drug can be used for your study