Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Summary

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Abstract

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

Introduction

The Open Field Maze (OFM) was initially developed in 1934 as a test to measure emotionality in rodents¹. It has attained the status of being one of the most widely used measures of behavior in animal psychology². It provides an easy and fairly rapid assessment of well-defined behaviors requiring no training to the test subject and little to no specialized training for the human administering the test. These attributes have led to wide-spread use of the open field maze in research extended to other animal species such as calves, pigs, rabbits, primates, honeybees and lobsters³. Part of its popularity arises from the fact that the psychological and physiological concepts underlying the tests are generally straight-forward and well understood. For example, it has been postulated that evolutionary forces have selected for a common response in animals such that most species display anxiety-mediated fear or flight responses to specific stimuli. Rodents for example, show distinct aversions to large, brightly lit, open and unknown environments⁴. We can assume they have been phylogenetically conditioned to see these types of environments as dangerous. All of these features are incorporated in the open field maze and form the basis of its use in behavioral paradigm testing.

An open field maze consists of a wall-enclosed area that is of sufficient height to prevent the subject from escaping. Typical maze shapes are circular or square with an area large enough, based on the size of the subject tested, to elicit a feeling of openness in the center of the maze. A number of variables can be scored in the open field maze with most parameters involving differing types of motor activity². Ambulation is the most common behavior studied but others such as latency or rearing can also be measured. Most often, rodent behavior is analyzed in a bare maze. However, the addition of objects, either one or many to the maze floor, adds the ability to see how the subject interacts with novel additional stimuli². Relevant parameters when objects are presented are typically the number of approaches to an object or in some cases, preference or aversion for one object over another.

Many behavioral tests of anxiety are based on the subject animal’s body activity and locomotion⁵. Interpreting behavioral tests for emotionality while separating non-emotional confounding factors, such as motor activity, has been the subject of intense debate^6,7. As the OFM was originally described, two measures of emotionality can be deduced, locomotor activity and fecal boli deposits or defecation¹. However, these two measures have been shown in some studies to be unrelated supporting the conclusion that emotionality in rodents is multidimensional⁵. Regardless, discrepancies in the literature regarding these measures and emotionality or anxiety in mouse models may be attributed to differences in analysis criteria or differences in testing procedures. Studies have conclusively linked results from OFM analysis with other measures of anxiety when comparing mouse models⁸.

Protocol

NOTE: All procedures performed here were submitted to and approved by IACUC (Office of Research Compliance) and were conducted following NIH guidelines. Mice used in the behavioral testing paradigm were naive and not used for other tests. The C57BL/6 Wild Type and Knock Out mice used in this protocol have been described previously⁹ and the data presented here are from that manuscript.

1. Preparation of the Testing Room and Open Field Apparatus

1. Use a multiple unit open field maze (OFM) consisting of four activity chambers was used for this analysis (Figure 1). Each chamber measured 50 cm (length) x 50 cm (width) x 38 cm (height) and was made from white high density and non-porous plastic.
2. Texture the floors of the maze for traction during ambulation while maze walls were smooth. Maze quadrants were completely empty for the purpose of this test. In consideration of the rest of this protocol, a single quadrant of the maze described above will be utilized to demonstrate the OFM.
3. Wipe the chamber with a 95% Ethanol prior to use and before subsequent tests to remove any scent clues left by the previous subject mouse.
4. Allow the ethanol to evaporate completely prior to testing mice. This may take 5-10 min between each testing session.
5. For this analysis, use the SMART Video Tracking software from PanLab/Harvard Apparatus to record and evaluate mouse movement.
   NOTE: Any commercial video tracking camera and software may be used to track the test subject and evaluate results from the open field maze. It is very important for the end user to understand how to calibrate and run the software used for each individual analysis. Regardless of the video camera and tracking software used, best results are obtained when the camera and software are correctly calibrated according to the manufacturer’s instructions.
6. Perform the testing in a standard lit room capable of containing the maze apparatus and the computer required to run the software. Suspend the video camera above the maze either by attaching it to the ceiling or by using any elevated support system which allows the camera lens to see the entire maze area (Figure 2).
7. As the human administrator of the test, be sure to have enough space in the room to be completely unobservable by the test subjects in the maze so as not to influence behavior of the mice.

2. Preparing the Software to Measure Activity

1. Open the Video Tracking software.
2. Once the software is opened, move the cursor to the “Single-subject tracking” option located under the “Data Acquisition” tab and single click to open this option.
3. Choose the “Static Background” option located at the bottom of the screen.
4. After choosing “Static Background” is chosen, it is necessary to use the software to take an image of the maze prior to addition of the test subjects. To do this, move the cursor to the “Photo” button located at the bottom of the screen and single click.
   NOTE: The software will take a picture of the scenario without the test subject which will be subtracted from the image taken during the tracking process. This results in only the movement of the subject being analyzed by the software.
5. Confirm that the background image taken above is completely removed by the tracking software by moving the cursor to the “Test” button located at the bottom of the screen and clicking once. A solid white field will be shown if the background image is completely removed from the tracking image. If lighting conditions change or the maze is accidentally moved, you will see black “shadows” in this field indicating the two images do not perfectly coincide. To remedy this situation, simply repeat step 2.4 above.
6. After confirming background settings, use the Timing option to configure the way time is controlled during acquisition. To do this, move the cursor to the “Configuration” tab and click once on the “Timings” heading. Use the newly opened window to enter experimental parameters.
7. For this protocol, chose the “Programmed time” option as 10 min tracking period. Set the “Latency period” to 5 sec to allow the user time to place the mouse in the center of the maze and move away prior to initiation of tracking. Enter the “Acquisition time” of 10 min for the duration of the test. Set the “Stop control” set to “When programmed time (10 min) is over” which will automatically turn off the camera and the tracking function of the software.
8. Move the cursor to the “Close” button after all timings have been set to close the window. You are now ready to begin the testing procedure.

3. Administration of the Open Field Test

NOTE: The software package used in this protocol allows the tracking of up to 16 individual mice at one time. For ease of completion and as mentioned above, the protocol discussed here is for a single mouse using a single quadrant of the OFM. For the equipment in use for this protocol, a maximum of 4 individual mice could be tracked using each quadrant of the maze. If utilizing a multi-enclosure maze, after placing the first subject mouse in its defined quadrant, place the remaining mice into their respective maze quadrant for tracking analysis. For the purposes of this protocol, further instruction will be specific to a single quadrant of the maze.

1. Bring the mice in their home cages from their housing room into the testing room. Allow the mice to acclimate to the procedure room for a minimum of 30 min prior to starting the test.
2. Remove a single mouse from the home cage by gently grasping its tail and place the mouse in the middle of the open field maze while concurrently activating the SMART software by single clicking on the Start button to begin tracking mouse movement. It is normal for the mouse to move immediately to the periphery walls of the maze and the timing of release and tracking capture of the mouse should coincide to record this movement.
3. Allow free and uninterrupted movement of the subject mouse throughout the respective quadrant of the maze for a single 10 min period during which time, the tracking software will record movement (Figure 3).
4. At the end of the test period, pick up the subject mouse gently, removing it from the maze and return it to its home cage.
5. Prior to cleaning the maze, visually count the fecal boli pellets present in the maze and manually record the numbers for further analysis.
6. Remove all fecal pellets and wipe up all spots of urination. Spray the floor and walls of the maze quadrant with 95% ethanol and wipe down with a clean paper towel. Allow the ethanol solution to completely dry prior to testing other mice.
7. Repeat the procedure with the next mouse.

4. Measurement and Analysis of Behavior During Testing Procedure

NOTE: For measurement, three aspects of open field behavior are readily characterized using this protocol (see discussion). A brief instruction on how to access these measurements in the video tracking software follows.

1. From the main screen of the SMART software, move the cursor to the “Zones” tab and single click “Definition” to open the Zone Editor.
2. Follow the detailed instructions in the SMART software User’s Manual do define zones or grids to overlay on the tracking paths. Here, the software was used to define a 5 x 5 grid of 10 cm squares covering the floor of the maze (Figure 4). Be sure to save the zone file created prior to closing the Zone Editor.
3. From the main screen of the SMART software, move the cursor to the “Analysis” tab and single click to open the Data Analysis window.
4. Move the cursor to the “File” tab and open the Zone file created above.
5. Move the cursor to the “Configuration” tab and open the “Track Analysis” option. This will open the “Single-subject analysis configuration” window.
   1. Move the cursor to the “Standard” tab and move the “Travelled distance” parameter from the Available Parameters box (left side) to the Included Parameters box (right side).
   2. Move the cursor to the “Zones Transitions” tab and move all appropriate parameters to the Included Parameters box as above.
   3. Make sure the “Full Track” box is checked at the bottom of the window.
   4. Move the cursor to the OK button and close the Single-subject analysis configuration window.
      NOTE: Depending on the analysis you wish to perform, many other options can be chosen in this window to mine data from the analysis. Read the detailed User’s Manual of your specific program to determine what parameters are most important for analysis of your data.
6. Under the File tab at the upper left of the program window, open the Single subject track window and place a check mark beside all tracks being analyzed. Move the cursor to the Check Mark button at the top of the window and click to close the Track Explorer window.
7. Move the cursor to the “Go” button in the Data Analysis window and single click to initiate analysis of the track data.
8. Analysis data can be output as either ASCII text files or it can be directly exported into an EXCEL spreadsheet. Use the output tools of the software program you are using to output the data for your own use.
   NOTE: Total distance traveled and time spent in indicated zones will be output following the data analysis steps outlined above. Again, it is stressed that the steps to reach these measurements represented here will differ depending on the user software used. But the data itself and the interpretation of the results should be similar independent of the software program used. It is also worth noting that test administrator bias is removed from this protocol as all the data collected is quantified data measured by the software and not the administrator. Thus there is no qualifiable element to the data collected as described.

Results

The average number of individuals per strain of mice tested in most cases is approximately 20 to generate sufficient statistical relevance. However, this number can be in the range of 8-30 depending on mouse availability. Depending on the measurement or comparisons required, it is also favorable to use age-matched subjects.

The first and arguably most important specific parameter to measure in the Open Field Maze is total ambulatory distance. While the unit of measure is irrelevant for compari...

Discussion

The Open Field Maze is one of the most widely used platforms in animal behavioral studies. A number of important conventional and ethological parameters^2,4 can be collected and analyzed during the performance of the OFM. These data allow the researcher to measure behaviors ranging from overall locomotor activity to anxiety-related emotional behaviors⁸. However, use of OFM is not without its shortcomings. One confounding issue is the wide range of static variables that can be manipulated during any t...

Materials

Name	Company	Catalog Number	Comments
Multi Unit Open Field Test	San Diego Instruments, Inc.	White 7001-0354	Any single or multi unit open field maze can be used
SMART DT Tracking Software	PanLab/Harvard Apparatus	76-0695	Any tracking software can be utilized with this protocol
Sony 990x Video Camera Recorder	Sony	CCD-TRV328	Any suitable video camera can be attached to computer for recording tracking profiles.