Name: Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice (Video) | JoVE Journal
Uploaded: 2015-02-06T15:00:00.000+00:00
Duration: 7 min 12 s
Description: Learn to assess locomotor activity and anxiety in mice using the Open Field Maze with video tracking software. This protocol enables precise measurement of emotional behaviors in rodent models.

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5 minutes

This work was supported by The National Institute of Health (NIH-2RO1NS033661) and by the Alabama Agricultural Experiment Station (HATCH ALA021-1-09017).

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Allow the ethanol to evaporate completely prior to testing mice. This may take 5-10 min between each testing session.</li>
	<li>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&#8217;s instructions.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
2. Preparing the Software to Measure Activity
<ol>
	<li>Open the Video Tracking software.</li>
	<li>Once the software is opened, move the cursor to the &#8220;Single-subject tracking&#8221; option located under the &#8220;Data Acquisition&#8221; tab and single click to open this option.</li>
	<li>Choose the &#8220;Static Background&#8221; option located at the bottom of the screen.</li>
	<li>After choosing &#8220;Static Background&#8221; 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 &#8220;Photo&#8221; 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.</li>
	<li>Confirm that the background image taken above is completely removed by the tracking software by moving the cursor to the &#8220;Test&#8221; 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 &#8220;shadows&#8221; in this field indicating the two images do not perfectly coincide. To remedy this situation, simply repeat step 2.4 above.</li>
	<li>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 &#8220;Configuration&#8221; tab and click once on the &#8220;Timings&#8221; heading. Use the newly opened window to enter experimental parameters.</li>
	<li>For this protocol, chose the &#8220;Programmed time&#8221; option as 10 min tracking period. Set the &#8220;Latency period&#8221; 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 &#8220;Acquisition time&#8221; of 10 min for the duration of the test. Set the &#8220;Stop control&#8221; set to &#8220;When programmed time (10 min) is over&#8221; which will automatically turn off the camera and the tracking function of the software.</li>
	<li>Move the cursor to the &#8220;Close&#8221; button after all timings have been set to close the window. You are now ready to begin the testing procedure.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Prior to cleaning the maze, visually count the fecal boli pellets present in the maze and manually record the numbers for further analysis.</li>
	<li>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.</li>
	<li>Repeat the procedure with the next mouse.</li>
</ol>
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.
<ol>
	<li>From the main screen of the SMART software, move the cursor to the &#8220;Zones&#8221; tab and single click &#8220;Definition&#8221; to open the Zone Editor.</li>
	<li>Follow the detailed instructions in the SMART software User&#8217;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.</li>
	<li>From the main screen of the SMART software, move the cursor to the &#8220;Analysis&#8221; tab and single click to open the Data Analysis window.</li>
	<li>Move the cursor to the &#8220;File&#8221; tab and open the Zone file created above.</li>
	<li>Move the cursor to the &#8220;Configuration&#8221; tab and open the &#8220;Track Analysis&#8221; option. This will open the &#8220;Single-subject analysis configuration&#8221; window.
	<ol>
		<li>Move the cursor to the &#8220;Standard&#8221; tab and move the &#8220;Travelled distance&#8221; parameter from the Available Parameters box (left side) to the Included Parameters box (right side).</li>
		<li>Move the cursor to the &#8220;Zones Transitions&#8221; tab and move all appropriate parameters to the Included Parameters box as above.</li>
		<li>Make sure the &#8220;Full Track&#8221; box is checked at the bottom of the window.</li>
		<li>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&#8217;s Manual of your specific program to determine what parameters are most important for analysis of your data.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Move the cursor to the &#8220;Go&#8221; button in the Data Analysis window and single click to initiate analysis of the track data.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

The Open Field Maze is one of the most widely used platforms in animal behavioral studies. A number of important conventional and ethological parameters2,4 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...

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&#8217;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 debate6,7. 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.

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

use of the open field maze to measure locomotor and anxiety-like behavior in mice

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

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.

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...

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

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 overall goal of this procedure is to introduce the open field maze as an easy to use platform to visualize general locomotor behavior and to specifically measure anxiety related emotional behaviors. In eroded model. This is accomplished by first setting up the maze platform and camera tracking system to measure the behaviors of interest.The second step is to introduce the mouse subject to the maze in such a way as to minimize background noise received by the camera. Next, the mouse is allowed free and uninterrupted movement throughout the maze en closure, which is captured by the camera using video tracking software. The final step is to analyze the data obtained from the tracking software to determine any differences in parameters used to measure anxiety related behaviors.Ultimately, the open field maze can be used to measure locomotor activity and anxiety related behaviors to gain insight into emotional behavior patterns in rodent models. One of the advantages of this technique over existing methods to measure anxiety in a rodent model like the elevated plus maze, is the number of ethological and conventional parameters that can be measured during a single testing session. To prepare the experiment, obtain an open field maze or OFM.This may consist of a single or multiple activity chamber maze. Each chamber should measure approximately 50 centimeters in length, 50 centimeters in width, and 38 centimeters in height, and should be made from high density and non-porous plastic floors of the maze should be textured for traction during ambulation and maze Walls should be smooth to inhibit climbing, leave the maze quadrants completely empty. For the purpose of this test, a single quadrant of an empty four quadrant maze will be utilized.To demonstrate the OFM prior to use, wipe the chamber with 95%ethanol to remove any scent clues left by the previous subject mouse. Allow the ethanol to evaporate completely for five to 10 minutes before testing the mice. Set up the testing in a normally lit room with light levels, approximately 500 to 1000 lux or normal office lighting conditions, and suspend a video camera above the maze by attaching it to the ceiling to enable the camera lens to see the entire maze area.Provide yourself enough space in the room so that you are unobservable by the test subjects in the maze. Otherwise, you may influence the mice's behavior. Open the video tracking software and select the data acquisition tab.Then the single subject tracking. Choose the static background option located at the bottom of the screen. Next, take a picture by clicking the photo button located at the bottom of the screen.Select the test button located at the bottom of the screen to confirm that the background image taken above is completely removed by the tracking software. A solid white field will be shown if the background image is completely removed from the tracking image. Black shadows will appear in this field if lighting conditions change or if the maze is accidentally moved, indicating the two images do not perfectly coincide.Remedy this error by repeating the calibration step. After confirming the background settings, configure the timing options by clicking the configuration tab. Once in the timing's heading, use the newly opened window to enter experimental parameters.Choose the 10 minutes tracking period in the programmed time option, and set the latency period to five seconds. Enter an acquisition time of 10 minutes for the duration of the test. Set the stop control to when programmed time is over, which will automatically turn off the camera and the tracking function of the software.Once all timings have been set, select the close button. Set the stop control to when the programmed time is over, which will automatically turn off the camera and the tracking function of the software. Once all timings have been set, select a close button to begin the open field test.Place home cages with mice from their housing room into the testing room. Allow the mice to acclimate to the procedure room for a minimum of 30 minutes prior to starting the test. Next, remove a single mouse from the home cage by gently grasping its tail.And place the mouse in the middle of the OFM while concurrently activating the smart software. Record the mouse immediately as it moves throughout the maze. Allow the mouse free and uninterrupted movement throughout the maze for a single 10 minute period.At the end of the test period, gently remove the mouse from the maze and return it to its home cage. Visually count the fecal boi pellets present in the maze and manually record the numbers for further analysis. Then 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. Repeat the procedure with the next mouse.The first and arguably most important specific parameter to measure in the open field maze is total ambulatory distance, wild type, or normal C 57 black six. Mice show similar ambulatory ability to a specific knockout, C seven black six mouse strain when total distance traversed is similar between strains. Further analysis of emotional behaviors is simplified because locomotor activity is removed from the equation as total ambulatory distance between the two mouse strains with similar Figma taxes or the tendency of a subject to remain close to walls was analyzed in the wild type versus knockout mice.Fig mota increases in the knockout mice in the outer zone, which represents a rise in anxiety levels. Knockout mice exhibited an increase in fecal bolli when compared to wild type mice. Increased number of BOI can be indicative of increased anxiety and emotionality of the subject animal.When performing this procedure, it's very important for the person administering the test to remain as still and quiet as possible, as any unnecessary noise or movement can influence the behavior of the subject being tested.

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Research

JoVE Journal

Behavior

The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Elevated Plus Maze for Mice

The plus-maze measures anxiety-like behaviour in rodents. There are two opposite closed and two opposite open arms; anxious rodents avoid the open arms. The central area is neither completely open nor closed, so time spent here is ambiguous and difficult to interpret. Here a modification of the plus-maze protocol eliminating this area is described.

The Successive Alleys Test of Anxiety in Mice and Rats

This video describes the open field behavior test used to examine general locomotion and exploration habits in...

Open Field Behavior Test: A Method to Assess General Locomotion and Exploration Habits

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Barnes Maze Testing Strategies with Small and Large Rodent Models

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Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

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Place and Response Learning in the Open-field Tower Maze

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Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

A comprehensive behavioral test battery of motor skills, mood—including social interaction, depression, and anxiety—and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Investigating Migraine-Like Behavior Using Light Aversion in Mice

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

Behavioral Characterization of an Angelman Syndrome Mouse Model

Learn to assess locomotor activity and anxiety in mice using the Open Field Maze with video tracking software. This protocol enables precise measurement of emotional behaviors in rodent models.

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

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