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. Figure 2.1 A rat in a square open field. 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 (Figure 2.2, top) 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 (Figure 2.2, bottom), 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.

Figure 2.2 Open field and novel tank diving test for zebrafish. Redrawn from

Kalueff et al. Trends in Pharmacological Sciences, 35: 63 - 75. 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. In the picture below, there is more background light in the arenas on the left than in the arenas on the right. 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 arena.

Figure 2.3 Minimize the shadows at the corners of open fields using even,

diffuse light, in any case not pointing to the floor since it would create shadows. 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.

We assume that you followed the procedure in arena settings on page

15.

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,

then draw the outline of the arena and select the number of sectors. Each sector is automatically defined as zone.

Zone group "Borders"

Check that the 'Wall' zone covers only the wall of the open field (see Figure 2.4). 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. Figure 2.4 Circular open field with 'Border' zone and 'Quadrants' zone. Figure 2.5 Rectangular open field with a subdivided arena.

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.

Default Trial Control

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.

We assume that you followed the procedure in detection settings on

page 17.

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. 6. Tracking starts after the subject is detected in the arena (see also Trial Control Settings on page 38). Make sure that tracking does not start because your arm is detected instead of the animal. To do so, for example, make a door in the open field, or increase the period that the subject needs to be detected before tracking starts (see page 38). If you use multiple arenas, tracking starts in each arena separately, depending on when the subject is detected in that arena. The subject is allowed to explore freely for a standardized time. Traditionally track duration was between 2 and 10 minutes, but video tracking with EthoVision XT allows longer tracking (see also page 38). 7. After the test has ended, take the subject out of the arena and put it back in its home cage. 8. Optionally, count the fecal boli in each zone of the open field. 9. Clean the open field and allow it to fully dry prior to introducing the next test subject.

Notes

tip When tracking white animals, then wear dark clothes and when

tracking dark animals then wear white clothes. This prevents EthoVision XT from detecting your arms while you release the animals in the arena. See also acquiring tracks on page 21.

score behaviors manually

Once you have defined your behaviors of interest in the Manual Scoring Settings (page 35), you can score those behaviors in two ways: Live. That is, during the test. On the Acquisition screen, click the Manual Scoring tab and score behaviors when these occur.

Offline. If you have recorded video, choose Acquisition > Manual

Scoring. Review the video and add or edit behavior scores.

For more information, see Acquire Data > Score behaviors manually in

the EthoVision XT Help.

Data Analysis

analysis profiles

The template experiment contains four analysis profiles:

Distance & Time - With the variables Distance Moved and Velocity,

the mean velocity and the total distance moved and the group means with their standard errors are calculated. The total distance moved is a measure of the activity of the test animal.

Movement - With this analysis profile the behavior of the animals

is divided into moving and not moving. The frequency, duration and latency to either of these states and the group means with their standard errors are calculated. Like total distance moved, movement is a measure for activity.

In zones - With this analysis profile, the frequency the animal

visited the zones defined in the arena settings and the group mean with its standard error are calculated. The data profile also contains the total time spent in the zones and the latency until the animal first enters either of the zones.

Behaviors - With this analysis profile, the frequency, duration and

the group means with their standard errors of the manually scored behaviors are calculated. In the template experiment, the behaviors rearing and grooming are predefined. Possibly, you added additional behaviors like defecating, or urinating (see page 35). You can add these behaviors to the analysis profile and calculate for example their frequency and duration.

analysis per zone

To calculate distance or speed in a specific zone

1. Choose Analysis > Data Profile > New.

Click on the Settings button on the Result box, select Results per

zone and subsequently select the zones you want to analyze. 2. Next, choose Analysis > Analysis Profile > open the analysis profile and select the variable (distance, velocity etc.). 3. Choose Analysis > Results > Statistics and Charts.

To calculate time spent in a specific zone

1. Choose Analysis > Analysis Profile > New. Choose In zone, select the zones you are interested in.
2. Choose Analysis > Results > Statistics and Charts.

time variation of behavior

Define Results per time bin. In this way, the dependent variables are calculated over each time interval. 1. Choose Analysis > Data Profile > New. 2. Click the Settings button on the Result box, select Results per time bin and specify the time interval. 3. Choose Analysis > Results > Statistics and Charts.

zone crossings

In addition to the predefined analysis profiles you can create an analysis profile for zone crossings. This way you can calculate the number of crossings from the border zone to the center zone. 1. Click the Add button next to Zone transition. 2. Select the zone sequence. 3. Depending on which option you select (Allow intermediate zone visits or Only count direct transitions) you can get different results.

For more information, see the Zone transition in the EthoVision XT

Help.

freezing behavior

If you want to quantify freezing behavior, which is a measure of the fear the animals experience, create an analysis profile for Activity state. Activity is defined as the amount of pixels in the arena that change at

any time. Click the Add button next to Activity state and define the

number of states and their thresholds (for freezing, the important threshold is Inactive below. On the Statistics tab, choose the statistics.

With this analysis profile the behavior of the animals is divided into the

states you specified.

Next, use the Plot Integrated Data function to visualize the activity

states. Adjust the settings in the Analysis profile until the state "Inactive" matches the freezing behavior of the animal in the video. The frequency, duration and latency to all those states is calculated in the next step (see Calculating statistics). For more information on Activity, see the EthoVision XT Help.

stretch-attend

Stretch-attend postures can be detected with the Body elongation

state variable in the Analysis profile.

For more information, see Body elongation state in the EthoVision XT

Help.

rat or mouse behavior recognition

To analyze the automatically detected behaviors, in the Analysis profile

under Rat or Mouse Behavior Recognition choose the behaviors you are

interested in. Furthermore:

If you want to analyze multiple behaviors as one, click Merged

Behavior. If you want to view the probability (that is, a measure of detection

uncertainty) associated with the behaviors detected, click Behavior

probability and specify the behaviors you are interested in.

comparing groups

1. Prerequisite: You can define groups of tracks based on independent variables (see page 19).
2. Choose Analysis > Data Profile > New.
3. Create a structure like that in the following picture.

To create a new branch, click the button next to Result and connect the

Start box with the new Result box.

visualizing data

Choose Analysis > Results > ... Figure 2.6 An example of data selection to compare two groups of tracks.

Plot tracks

You can view your tracks on a still image of the background. Tracks can be shown in different colors according to the values of independent variables (for example, blue for animals treated with saline and red for drug-treated animals). Sample points can be shown in different colors according to the values of dependent variables, for example red when the animal was moving fast.

Plot heatmaps

You can make heatmaps of the location of the subject.

Plot integrated data

You can look at a track with the video file in the background. When you plot integrated data you can also view Time Event plots of your independent variables.

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Source: EthoVision XT 17.5 Application Manual, The Open Field Test