Open-field test v2 01/Nov/2008 RIKENMPP_007_002 test 0 1 testSt test modification

The purpose of open-field tests is to examine mouse responses to new and unfamiliar environments (novel environments). A novel environment causes mice to move around and explore, which is accompanied by increased locomotion. Intense anxiety and fear, however, may cause mice to crouch, freeze, and suppress locomotion. Thus, open-field tests often adopt the amount of locomotion (or suppression of locomotor activity) as a measure of anxiety and fear. In general, mice that do not show locomotor activity and defecate more often in an open-field test are assumed to be experiencing intense anxiety and fear, whereas mice that move around and defecate less often are thought to feel less anxiety and fear. Other measures of anxiety used in open-field tests include the amount of time spent near the walls (thigmotaxis) and stretching (standing still or walking slowly with a low, stretched posture). Measures of decreased anxiety include frequency of rearing (standing on the hind legs and looking around) and grooming (e.g., face washing).

These procedures can be used with the mouse phenotyping platform in RIKEN BRC. The tests should be performed by trained staff members who understand the safety and hygiene rules required by RIKEN. Any queries, comments, or suggestions, relating either to this SOP in general or to a specific problem encountered during a procedure, should be addressed to the Behavioral Neuroscience Unit Leader. Any deviation from this protocol must be reported to the Behavioral Neuroscience Unit Leader.

Testing is carried out in accordance with common animal handling procedures. Coveralls (in the rearing facility) or laboratory coats (in the laboratory facility) must be worn with gloves at all times in the work area. If animals are dropped on the floor, they should be treated in accordance with the safety and hygiene guidelines of the rearing facility at RIKEN. If something unexpected happens during the primary screening, it must be quickly reported to the Behavioral Neuroscience Unit Leader and recorded.

Mice that do not show locomotor activity and those that excrete a large amount of feces are commonly thought to be experiencing intense anxiety and fear, whereas mice that are actively locomoting with infrequent defecation are not displaying anxiety and fear. In addition to anxiety and fear, however, various factors, such as general activity and the inclination to explore, can influence mouse locomotion in an open-field. Therefore, interpretation of the test results must take these factors into account. Summarized below are important points to keep in mind when interpreting the results of an open-field test. Interpreting the results of an open-field test from the viewpoint of anxiety and fear requires that the novel environment caused the decreased or increased locomotion. Decreased activity by a mouse in an open-field test may simply reflect a general low degree of activity for that mouse, instead of a freezing response resulting from anxiety and fear. When irregular levels of locomotion are observed in an open-field test, it is advisable to check the general activity level of the mouse in its home cage, in which the mouse is unlikely to be markedly affected by anxiety and fear. Although open-field tests assume locomotion is suppressed by anxiety and fear, these responses may lead to escaping activity and increased locomotion. When a mouse shows greater activity or looks restless as it runs around in an open-field, it is advisable to place the mouse in a new, unfamiliar scene that includes a place for the mouse to hide; examples include the Light/Dark Transition Test and the elevated plus maze (see reference 1). If the mouse shows decreased activity in an environment with a hiding place, the previously observed increase in locomotion was likely a result of increased escaping activity. The level of anxiety and fear may decrease with time. Thus, it is important to record time-dependent changes in behavior. When the amount of locomotion is consistently low, the mice may be displaying decreased general activity and a reduced inclination to explore. If the amount of locomotion is selectively suppressed immediately after the mouse is placed in a test apparatus, however, the suppression likely results from anxiety and fear.

Light illumination tends to decrease the amount of locomotion in an open-field. Because a difference between a test group and a control group in their sensitivities to illumination is likely to result in different amounts of locomotion, one should pay attention to the influence of vision, particularly when the eye colors of the groups differ. Open-field tests examine responses to a novel environment. Repeating a test will compromise the novelty of the test scene. Because results vary significantly between the first and second tests, data should always be grouped for analysis based on the test number when the test has been repeated. Because rearing conditions (individual or group rearing) and past experimental history also can influence the data, the test and control groups should be raised similarly and share the same experiences.

Our laboratory uses an open-field made of white vinyl chloride: 400 mm in length, 400 mm in width, and 320 mm in height (O'Hara and Co.). The ceiling is open and the floor is removable tray, which is easy to clean. Although there are no size restrictions for the open-field, a relatively large, open box is required to produce anxiety and fear in the mouse. Many laboratories use a round box or a square box (enclosure) with sides 400-900 mm long. To monitor and record the mouse movements during an experiment, a CCD camera, a TV monitor, and a video recorder are used. The CCD camera is mounted above the center of the open-field using a camera angle or a camera support. Our laboratory mounts a 4-W fluorescent lamp above the center of the open-field at approximately the same position as the CCD camera, so the experiments can be performed with the ceiling lamp turned off (intensity of illumination, 90 lux on the floor). Because mice prefer a sheltered area in an open-field, the field should be illuminated such that there is no shadow in the open-field.

1000-fold diluted Purelox (6% sodium hypochlorite) solution Paper towel Duster

Transfer a mouse from a rearing room to the testing room 15 minutes before starting the experiment (note 1). (note 1) Take care not to stimulate the mouse before starting the experiment. Do not change the cage on the day of the experiment, because it may influence the test result significantly. Soak the floor cloth in the drug solution, wring it out, and wipe the floor and walls of the apparatus clean. Hold the mouse by the tail and gently place it in a corner of the open-field. Leave the mouse in the apparatus and measure the amount of locomotion every minute for 20 minutes (note 2). (note 2) Five minutes is sufficient as a testing time, because the period immediately after the mouse is placed in the apparatus is the most important for analysis of anxiety- and fear-related behavior. Our laboratory uses a longer testing time to observe not only anxiety and fear but also general activity and familiarization with the test environment. Record the mouse behavior on video or observe it while watching the TV monitor (note 3). (note 3) Directly observing the open-field during the experiment may cause the mouse to freeze in response to the human. Therefore, the mouse should be observed via the TV monitor during the experiment. Take the mouse out of the apparatus after 20 minutes. Remove any feces with a paper towel and clean the floor (tray) with water.

Crawley, J.N. Emotional behaviors: animal models of psychiatric diseases. What's wrong with my mouse? Behavioral phenotyping of transgenic and knockout mice. Wiley-Liss. 2000: 179-206 Tests of animal behavior functions–bridging the individual level to a molecular level. Seitai No Kagaku, 45 (5), 1994 (translation). Martin, P. and Bateson, P. Measuring Behaviour: An Introductory Guide. Cambridge University Press, 1993. Archer, J. Tests for emotionality in rats and mice: A review. Animal Behaviour. 1973;21:205-235. Takahashi, A. et al. Multivariate analysis of temporal descriptions of open-field behavior in wild-derived mouse strains. Behavior Genetics. 2006; 36:1-12.