Protocol for Open Field Test (OF)
Materials Required
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Principle
The Open Field Test is a rodent behavioral assay that measures spontaneous locomotion, exploratory behavior, and anxiety-like behavior when an animal is placed in a novel open arena. The main readouts are total distance traveled, movement time, velocity, center-zone entries, center-zone time, peripheral-zone time, and thigmotaxis[1][2][3][4][5].
The assay is based on the conflict between exploration of a novel environment and avoidance of exposed open areas; higher center exploration is commonly interpreted as lower anxiety-like behavior, whereas increased wall-following or peripheral occupancy is interpreted as higher anxiety-like behavior[4][5][6][7].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Vehicle solution is used as the negative control when pharmacological treatment is tested[4][5].
• Known anxiolytic drugs, such as benzodiazepines, may be used as positive controls when the experiment is designed to validate anxiolytic-like effects[5][6][8].
• Mice or rats are used depending on the study design; species, strain, sex, age, and prior handling must be reported because these variables influence locomotor and anxiety-like measures[2][3][4][9][10].
• Known anxiolytic drugs, such as benzodiazepines, may be used as positive controls when the experiment is designed to validate anxiolytic-like effects[5][6][8].
• Mice or rats are used depending on the study design; species, strain, sex, age, and prior handling must be reported because these variables influence locomotor and anxiety-like measures[2][3][4][9][10].
Equipment and instruments
• Use an open-field arena with surrounding walls, video camera or infrared-beam activity monitoring system, tracking software or blinded manual scoring method, timer, and cleaning materials for the apparatus between animals[1][2][3].Controls
• Use vehicle-treated controls for drug studies, untreated or wild-type controls for genotype/model studies, and positive anxiolytic controls when validating anxiety-like behavior; locomotor outcomes must be analyzed alongside center-zone outcomes because altered activity can confound anxiety interpretation[3][4][5][8].Experimental Procedure
Preparation Steps
• Acclimate animals to the testing room before testing and keep lighting, noise, arena size, floor material, test duration, time of day, and handling consistent across groups[1][2][3][4].• Prepare the open-field arena and define center and peripheral zones before testing; one published mouse protocol used a 42 × 42 × 42 cm polyvinyl chloride box with camera tracking of movement in central and peripheral zones[1].
• Clean the arena between animals using the same method across all subjects to reduce odor-related carryover effects[2][3].
• Randomize testing order and blind the scorer or analyst to treatment or genotype when feasible[2][3].
Operation Steps
• Place the animal in the open-field arena according to the predefined protocol, start recording immediately, and allow free exploration for the selected test duration[1][2][3].• Record locomotor activity using video tracking, infrared-beam breaks, or validated manual scoring; common outputs include total distance traveled, velocity, movement duration, resting duration, zone entries, and time spent in center versus periphery[1][2][3].
• Score anxiety-like behavior using center-zone exploration and thigmotaxis measures, while interpreting these data together with total locomotion to distinguish reduced anxiety-like behavior from nonspecific hyperactivity or hypoactivity[4][5][6][7].
• After testing, remove the animal and return it to the home cage, then clean the arena before the next animal[2][3]• Data Acquisition and Analysis
Primary locomotor endpoints include total distance traveled, movement time, velocity, and beam breaks or tracked path length; primary anxiety-like endpoints include center-zone time, center-zone entries, peripheral-zone time, and thigmotaxis[1][2][3][5][7].
• Interpret center-zone behavior only alongside locomotor activity, because increased or decreased movement can independently change center entries and center time[3][4][5].
• For drug studies, compare treatment groups with vehicle controls and include an anxiolytic positive control when validating the assay; benzodiazepine-sensitive exploratory measures have been reported in open-field-related anxiety paradigms[5][6][8].
• For animal-model or genotype studies, use the individual animal as the biological replicate and report strain, sex, age, group size, arena dimensions, lighting, test duration, zone definitions, tracking method, blinding, exclusion criteria, and statistical test[2][3][4][9].
Troubleshooting
Problem: Center-zone time changes, but total locomotion also changes.
Possible Cause: The treatment or genotype may alter motor activity rather than anxiety-like behavior.Literature-supported Solution: Analyze total distance, velocity, and movement time together with center-zone measures, and confirm interpretation with complementary anxiety assays[3][4][5].
Problem: Results differ strongly between mouse strains.
Possible Cause: Baseline locomotor and anxiety-like behaviors vary by genetic background.Literature-supported Solution: Use one strain within an experiment, include strain-matched controls, and avoid comparing across strains without appropriate controls[4][9][10].
Problem: High variability occurs across testing days or cohorts.
Possible Cause: Open-field behavior is sensitive to apparatus, lighting, novelty, handling, and procedural differences.Literature-supported Solution: Standardize arena dimensions, illumination, handling, testing duration, and analysis parameters, and report them fully[1][2][3][4].
Problem: Anxiety-like interpretation is unclear.
Possible Cause: The Open Field Test measures multiple interacting behaviors, including locomotion, exploration, and avoidance of open areas.Literature-supported Solution: Use complementary assays such as elevated plus maze or light-dark box rather than relying on OF alone[4][5][9].
References:
- [1]. Kraeuter AK, et al. The Open Field Test for measuring locomotor activity and anxiety-like behavior. Methods Mol Biol. 2019;1916:99-103. [Content Brief]
- [2]. Seibenhener ML, et al. Use of the open field maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp. 2015;(96):e52434. [Content Brief]
- [3]. Tatem KS, et al. Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases. J Vis Exp. 2014;(91):e51785. [Content Brief]
- [4]. Walsh RN, et al. The Open-Field Test: a critical review. Psychol Bull. 1976;83(3):482-504. [Content Brief]
- [5]. Prut L, et al. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol. 2003;463(1-3):3-33. [Content Brief]
- [6]. Crawley JN, et al. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav. 1980;13(2):167-170. [Content Brief]
- [7]. Simon P, et al. Thigmotaxis as an index of anxiety in mice. Influence of dopaminergic transmissions. Behav Brain Res. 1994;61(1):59-64. [Content Brief]
- [8]. Choleris E, et al. A detailed ethological analysis of the mouse open field test: effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field. Neurosci Biobehav Rev. 2001;25(3):235-260. [Content Brief]
- [9]. 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]
- [10]. van Gaalen MM, et al. Behavioural analysis of four mouse strains in an anxiety test battery. Behav Brain Res. 2000;115(1):95-106. [Content Brief]
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