Protocol for Elevated Plus Maze Test
Materials Required
Principle
The Elevated Plus Maze is a rodent anxiety-like behavior assay based on the conflict between spontaneous exploration and avoidance of open, elevated, exposed spaces. The apparatus contains two open arms and two closed arms arranged in a plus shape, and rodents normally spend more time in closed arms than open arms[1][2][3][4].
The assay readout is generated by recording arm entries, time spent in open and closed arms, and related exploratory behaviors. Increased open-arm time or open-arm entries is commonly interpreted as reduced anxiety-like behavior, whereas reduced open-arm exploration is interpreted as increased anxiety-like behavior[1][2][3][4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
Reagents and chemicals
• Known anxiolytic drugs, especially benzodiazepines such as chlordiazepoxide or diazepam, may be used as positive controls because they increase open-arm exploration in validated EPM studies[2][3][4][6].•
Mice or rats are used depending on the study design; species, strain, age, sex, housing, and prior testing history should be reported because these variables affect EPM behavior[1][4][7][8].
Equipment and instruments
• Use an elevated plus-shaped maze with two open arms, two closed arms, and a central platform, plus a video camera, video-tracking software or blinded manual scoring system, timer, and cleaning materials for use between animals[1][4][5].Controls
• Use vehicle-treated controls for drug studies, genotype- or disease-model matched controls for biological studies, and an anxiolytic positive control when validating the assay.• Include locomotor measures such as total arm entries because altered activity can confound anxiety-like interpretation[1][2][3][4][6].
Experimental Procedure
Preparation Steps
• Place the maze in a quiet test room and keep illumination, room cues, maze height, arm dimensions, cleaning procedure, handling, and testing time consistent across groups[1][4][7].• Acclimate animals to the testing room before testing, randomize test order, and blind the observer or analyst to treatment or genotype when feasible[1][4].
• Clean the maze between animals using the same procedure across all groups to reduce odor-related carryover effects[1][4].
Operation Steps
• Place the mouse or rat on the central platform at the junction of the four arms, commonly facing an open arm, and begin recording immediately[1][4].• Allow the animal to freely explore the maze for the selected test duration; a 5-min session is widely reported in protocol and validation studies, although some mouse protocols record for 10 min to increase opportunity to detect phenotypes[1][4][5].
• Record open-arm entries, closed-arm entries, time spent in open arms, time spent in closed arms, center time, total entries, and, when used, ethological measures such as head dipping, stretch-attend posture, and risk-assessment behavior[1][3][5][6].
• After testing, remove the animal from the maze, return it to the home cage, and clean the apparatus before the next subject[1][4]• Data Acquisition and Analysis
Primary anxiety-like outcomes are percentage of time spent in open arms and percentage of open-arm entries.
Total arm entries or closed-arm entries should be analyzed as locomotor or general activity measures to identify motor confounds[1][2][3][4][6].
• Interpret increased open-arm exploration as reduced anxiety-like behavior only when locomotor activity is not independently increased or suppressed[1][3][6].
• Use the individual animal as the biological replicate and report species, strain, sex, age, group size, maze dimensions, maze height, illumination, test duration, scoring definition for arm entry, tracking method, blinding, exclusion criteria, and statistical test[1][4][7][8].
• Avoid repeated EPM testing unless the design specifically addresses retesting, because prior EPM exposure can change open-arm behavior and drug sensitivity[6][9].
Troubleshooting
Problem: Open-arm time changes but total arm entries also change.
Possible Cause: The treatment or genotype may alter locomotor activity rather than anxiety-like behavior.Literature-supported Solution: Analyze total entries or closed-arm entries together with open-arm measures before interpreting anxiolytic-like or anxiogenic-like effects[1][3][6].
Problem: Results vary across cohorts or laboratories.
Possible Cause: EPM behavior is sensitive to procedural variables such as lighting, apparatus dimensions, strain, age, sex, housing, and testing conditions.Literature-supported Solution: Standardize these variables and report them in detail[1][4][7][8].
Problem: Drug effects are reduced or absent during repeat testing.
Possible Cause: Prior exposure to the EPM can produce a one-trial tolerance or retest effect.Literature-supported Solution: Avoid repeat testing when possible, or use a retest design specifically developed to address prior exposure effects[6][9].
Problem: Anxiety-like interpretation is uncertain.
Possible Cause: EPM reflects multiple behaviors, including exploration, avoidance, risk assessment, and locomotion.Literature-supported Solution: Include ethological measures and complementary anxiety assays rather than relying only on open-arm time[3][5][6].
Verweise:
- [1]. Walf AA, et al. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc. 2007;2(2):322-328. [Content Brief]
- [2]. Pellow S, et al. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods. 1985;14(3):149-167. [Content Brief]
- [3]. Rodgers RJ, et al. Anxiety, defence and the elevated plus-maze. Neurosci Biobehav Rev. 1997;21(6):801-810. [Content Brief]
- [4]. Kraeuter AK, et al. The Elevated Plus Maze Test for measuring anxiety-like behavior in rodents. Methods Mol Biol. 2019;1916:69-74. [Content Brief]
- [5]. Komada M, et al. Elevated Plus Maze for mice. J Vis Exp. 2008;(22):1088. [Content Brief]
- [6]. Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berl). 1987;92(2):180-185. [Content Brief]
- [7]. Carola V, et al. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav Brain Res. 2002;134(1-2):49-57. [Content Brief]
- [8]. Albani SH, et al. Behavior in the elevated plus maze is differentially affected by testing conditions in rats under and over three weeks of age. Front Behav Neurosci. 2015;9:31.
- [9]. Schneider P, et al. A novel elevated plus-maze procedure to avoid the one-trial tolerance problem. Front Behav Neurosci. 2011;5:43. [Content Brief]
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