Knowledge Base

Introduction

The Open Field for Rodents

The open field test was first published 1932 by C. S. Hall and E.L. Ballachey to determine the fearfulness of rats. Today, the test is often used with rats and mice to investigate their locomotory behavior and their willingness to explore. Also, the test is one of the most commonly used methods to investigate anxiety in rats and mice. It is based on the natural tendency of rats and mice to spend more time in the corners and the periphery than in the center. Changes in these measures are often used to assess the sedative or stimulant effects of pharmacological agents.

The test arena consists of a circular or rectangular empty open space with walls to prevent the animals from escaping. The arena is divided into a border zone and a center zone. In addition, the arena can be sub-divided into a number of other zones, like north, east, south and west zones, or corner zones.

Common behaviors to be measured are:

• Time spent in the border zone and center.
• Time until the border of the center zone is first crossed.
• Walking distance in each of the zones.
• Percentage of time the animal is mobile/immobile.
• Walking speed in each of the zones.
• Frequency of rearing.

References:

• Hall, C., Ballachey, E. L., 1932. A study of the rat's behavior in a field. A contribution to method in comparative psychology. University of California Publications in Psychology, Vol 6, 1-12.
• Gould, T.D., Dao, D.T., Kovacsics, C.E., 2010. The Open Field Test. Neuromethods, Vol 42, 1-20.
• De Visser, L., Van Den Bos, R., Kuurman, W.W., Kas, M.J.H., Spruijt, B.M., 2006. Novel approach to the behavioural characterization of inbred mice: automated home cage observations. Genes brain and behavior, Vol 5(6), 458-466.

For the complete list of publications, see the results in Google Scholar.

On the downloads section of the Noldus website, my.noldus.com, you find a number of sample experiments, including the open field.

The Open Field for Zebrafish

Open Field

The open field test is used to evaluate exploratory and affective behaviors. Fish are filmed with a video camera positioned overhead. The bottom of the tank is divided into two virtual zones, center and periphery (a circular zone with inner boundary 5 cm from the testing tank wall), which enables quantification of regional activity.

In addition to standard movement parameters, primary behavioral measures included the distance traveled, number of visits and time spent in central and peripheral zones, and the ratio of time spent in the center zone over total testing time.

Novel Tank Diving Test

The novel tank test is used to assess exploratory and affective domains. The novel tank is a trapezoidal tank maximally filled with treated water and divided into two equal, horizontal regions (top/upper and bottom/lower zones).

Primary behavioral measures include the number of visits and time spent in central and peripheral zones, the ratio of time spent in the top zone over total testing time, latency to enter the top zone, time spent in the top zone, number of transitions to top zone.

Similar to behavioral tests used in mammalian models, affective phenotypes are inferred from modulation of thigmotaxis (center/periphery in the open field test) and geotaxis (top/bottom in the novel tank diving test).

References:

• Kalueff A.V., A.M. Stewart, R. Gerlai (2014). Zebrafish as an emerging model for studying complex brain disorders. Trends in Pharmacological Sciences, Vol. 35 (2), 63-75.

Example videos: https://www.youtube.com/watch?v=7vhK8fq_W4A

Physical Setup

The following suggestions are meant to optimize video tracking:

• The camera should have a good view of the entire region the animal can be in. The arena should fill the field of view.
• The lighting should be as even as possible throughout the arena. Make sure there are no shadows in the corners. Bright lights are not necessary but should you need to supplement the light, consider red (or infra-red) light, as the animals will be less sleepy. Regardless of the light level chosen, the investigator should use a light meter to record the lighting level and report this level in any publication.
• When working with multiple arenas, make sure that the background light intensity is the same across arenas. This is usually not a problem, as long as the contrast between each animal and its arena background is much bigger than the difference in brightness between arenas.
• The lighting should be diffuse, so as not to cast strong shadows (which might be tracked instead of the animal). Even, diffuse lighting is important to optimize tracking of the nose.
• The background should contrast with the animal. If necessary use a different setup for light and dark colored animals. Matt, gray walls often give good results.
• Depending on circumstances, pools of urine can cause problems with the tracking because they reflect light. An absorbent base can help.
• Make sure the sides of the box are not reflective (e.g. sanding). Alternatively, exclude the sides from the arena, but make sure that the animal is tracked well in the entire border zone.
• Always place the apparatus in the same position in the room, cues from overhead might influence the behavior of the animal. For the same reason the experimenter should not be visible to the animal during the trial (automated tracking makes this possible).
• Minimize disturbance from audio sources, especially from ultrasound sources such as ventilation fans, CRT monitors, or strip-lights. You can mask audio disturbance by using the same background sounds, for example of a radio, in the test room as in the room where the animals are normally housed.
• When you handle the animals, be careful to minimize stress.
• If you use multiple arenas, make sure that the animals in the arena do not influence each other. The animals should, for example, not be able to hear or smell each other.
• In EthoVision XT, choose Help > Video Tutorial and watch the video chapter Set Up Your Test Environment. That provides useful tips about the camera position, how to improve lighting and contrast with the background.
• If you intend to use the Deep learning method to track the subject's nose point, note that there are additional requirements and limitations. See the EthoVision XT Help.

The Open Field Experiment in EthoVision XT

Create an experiment. For details, see Chapter 1 of this manual, or in EthoVision XT press F1 and see Setup an Experiment in the EthoVision XT Help.

Experiment Settings

Choose Setup > Experiment Settings.

• To detect freezing behavior, select Activity analysis under Analysis options. Click the video button next to it and watch the video tutorial to learn about Activity analysis.
• If you work with rats or mice, you can let EthoVision XT automatically detect behaviors like Rearing, Grooming, and Sniffing. Select Behavior recognition under Analysis options and choose the options that apply to your experimental setup. The Behavior recognition option is only available if you have the Rat Behavior Recognition module or the Mouse Behavior Recognition module of EthoVision XT.

Manual Scoring Settings

Choose Setup > Manual Scoring Settings.

If you used the template for an open field experiment, two start-stop behaviors have been predefined in the manual scoring settings. These are the behaviors rearing and grooming. Depending on your research question, you can add other behaviors that can be manually scored, like defecating, which is a measure for anxiety, and urinating and vomiting.

Note that with the Rat (or Mouse) Behavior Recognition module you can have EthoVision XT detect a number of behaviors automatically.

For more information, see Manual Scoring Settings and Behavior Recognition in the EthoVision XT Help.

Arena Settings

Choose Setup > Arena Settings > Open Arena Settings 1.

Arena

Check that the arena covers the whole area in which you want to track the animal. Remember to include enough of the walls so that the animal is still tracked when it rears, but exclude any bright reflective rims that might interfere with tracking. Make sure the label stays inside the arena.

Tip: If you create an experiment from scratch, the arena is not yet defined. Click 2. Select Shape and Draw Arena and choose the drawing tool. If you want to have the open field divided in quadrants, choose one of the subdivided tools. Depending on the shape of your open field, draw the outline of the arena and select the number of sectors. Each sector is automatically defined as a zone.

Zone Group "Borders"

Check that the Wall zone covers only the wall of the open field. If necessary, adjust the center zone and the border zone.

Zone Group "Quadrants"

Check that the zones NW, NE, SE, and SW divide the Floor zone in four equal quarters.

Zone Group "Squares"

Check that the zones divide the arena in equal squares.

Arena Settings for Rat/Mouse Behavior Recognition

When you selected Behavior recognition in the Experiment Settings (only available when you have the Rat or Mouse Behavior Recognition Module of EthoVision XT), defining a few special zones and points significantly increases behavior recognition accuracy.

• Feeder zones. To detect eating at the feeder.
• Drinking spout points. To detect drinking.
• Wall zones. To detect rearing to wall and to discriminate this from unsupported rearing.

For the exact procedure, see Zones and points for Behavior recognition in the EthoVision XT Help.

Trial Control Settings

In the Trial Control Settings you can define conditions for the start and stop of the data acquisition.

Choose Setup > Trial Control Settings.

The templates for the open field contain two Trial Control Settings profiles:

• Default.
• Track duration 10 (or 30) min - When you use this profile, tracking starts automatically 2 seconds after the animal has been placed in the arena. The track stops automatically after 10 or 30 minutes.

If you use Behavior Recognition or Activity Analysis, the default Trial Control rule is slightly different. See Trial Control in the EthoVision XT Help.

To stop acquisition after some specific time, open a Trial Control Settings profile, click the Settings button in the Condition - Time (1) box, and set the time you require.

Traditionally, the open field test had a duration of between 2 and 10 minutes. This short length of time was chosen in the past because the data were acquired manually. It also emphasizes the exploratory behavior and response to novelty, rather than baseline activity of the rodents. Technology like video tracking with EthoVision XT allows for much longer periods of monitoring.

Detection Settings

Choose Setup > Detection Settings.

Sample Rate

Under Video, set the sample rate. Common values are, expressed in number of images analyzed per second:

• For rats: 5 samples/s.
• For mice: 12.5 samples/s.
• For nose-tail tracking of all rodents: 25 to 30 samples/s.
• For adult zebrafish: 5 samples/s.

Remember, however, that the optimal sample rate is much depending on a variety of factors, including the characteristics of the setup, and even the animal's strain and behavior. Faster animals need a higher sample rate. This is also valid for fish. If the fish shows very rapid movements, then set the sample rate to the maximum; in other cases a sample rate of 5 fps may be enough.

To Detect Freezing Behavior

Open the Activity section in the Detection Settings pane. This option is only available if you selected Activity analysis under Analysis options in the Experiment settings. Enter the Activity threshold for detecting a change in the pixels in the arena. Start with the default value and check the purple-colored pixels in the video image. Adjust the threshold in such a way that when the animal is completely still, those pixels should be almost completely absent.

Rat or Mouse Behavior Recognition

If you selected Behavior Recognition in the Experiment settings (only available when you have the Rat or Mouse Behavior Recognition module of EthoVision XT), remove the tail from the detected body contour. To do so, in the Detection Settings pane:

1. Under Subject Contour, select one or more pixels for the first Erosion field until the tail is no longer detected. The detected body area (in yellow) looks now smaller.
2. Increase the Dilation filter until the detected body area is the same as before.

Next, open the Behavior section in the Detection Settings pane. Wait until the rat has a normal, straight, posture (so not bending or rearing) and is moving forward. You should see the hips of the rat moving. Click Grab and check that the body contour is correct. You can also enter the values manually if you know them from previous experiments. However, in this case make sure that the conditions are equal, like lighting, size of the animal and distance between the animal and the camera. The detected body must have a minimum length of 60 pixels (sum of nose-point to center-point and center-point to tail-base) for Behavior Recognition. The detected body must also be smaller than half the arena size.

For more information, see the EthoVision XT Help.

Acquiring Data

Protocol

A typical open field test is carried out in the following way:

1. Transport the subject to the testing room at least 1 hr. prior to testing, to allow them to acclimate to the experimental room. This time period should be extended if the transfer involves excessive changes in the environment.
2. Clean the open field, even if it is not dirty prior to the initial trial. This way all animals experience the residual smell of the disinfectant equally.
3. In EthoVision XT, click the Start Trial button. EthoVision XT waits until the subject is detected in the arena.
4. Place the subject in the open field. Commonly, the subject is placed in the center. However, placing adjacent to a wall is sometimes used to avoid immediate exposure to a stressful situation. Also, in case of a large open field, the position adjacent to the wall may be easier to reach.
5. The researcher should leave the room, or position himself or herself as far as possible away from the arena and field of view of the subject. Also the tester should remain quiet throughout each trial.

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Source: EthoVision XT 19 - Application Manual