Source Paper
Elevated Plus Maze for Mice
Munekazu Komada, Keizo Takao, Tsuyoshi Miyakawa
Journal of Visualized Experiments • 2008
Source Paper
Munekazu Komada, Keizo Takao, Tsuyoshi Miyakawa
Journal of Visualized Experiments • 2008
Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing. Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.
Objective: Measure anxiety-like behavior in mice based on their aversion to open and elevated areas and natural exploratory behavior
This is a Elevated Plus Maze protocol using mouse as the model organism. The procedure involves 9 procedural steps, 4 equipment items, 1 materials. Extracted from a 2008 paper published in Journal of Visualized Experiments.
Model and subjects
mouse • Not specified in protocol; C57BL/6J used for reference data • Not specified • Not specified • Not specified • Not specified per trial; reference data from n=914 C57BL/6J mice
Study window
~1.6 hours hands-on
Core workflow
Animal housing and light cycle • Pre-testing habituation • Practice trial with test animal
Primary readouts
Key equipment and reagents
Verified items
0
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Mice are housed with a 12-hour light/dark cycle with lights on at 7:00 AM
Note: Standard housing conditions maintained throughout study
“Mice are housed with a 12-h light/dark cycle (lights on at 7:00 AM), as previously described”
All experimental mice are transferred to the behavior testing room 30 minutes prior to beginning the first trial to habituate to the condition of the behavior testing room
Note: Allows mice to acclimate to testing environment
“All the experimental mice are transferred to the behavior testing room 30 min prior to beginning the first trial to habituate to the condition of the behavior testing room”
A practice trial is conducted using a practice animal to ensure recording system is functioning properly and to maintain uniform testing conditions by providing prior noise and odor cues for subsequent test animals
Note: The first mouse in entire session would experience different conditions without this practice trial
“There are two purposes of the test trial using a practice animal. One is to make sure that everything is fine with the recording system. Another is to keep the testing condition as uniform as possible”
A mouse is placed in the center area of the maze with its head directed toward a closed arm
Note: Standardized starting position and orientation
“A mouse is placed in the center area of the maze with its head directed toward a closed arm”
The elevated plus maze test is recorded using a video camera attached to a computer. Mice are allowed to move freely about the maze for 10 minutes. The number of entries into each arm and the time spent in the open arms are recorded
Note: 10 min recording used instead of common 5 min to increase opportunity to detect phenotype
“Mice are allowed to move freely about the maze for 10 min. The elevated plus maze test is recorded using a video camera attached to a computer, which is controlled by a remote device. The number of entries (an entry is defined as the center of mass of the mouse enters the arm) into each arm and the time spent in the open arms are recorded”
The Image EP program automatically calculates distance traveled, number of entries into each arm, time spent in each arm, and percent of entries into the open arms
Note: Image EP is based on public domain Image J program modified by Tsuyoshi Miyakawa
“The distance traveled, the number of entries into each arm, the time spent in each arm, and the percent of entries into the open arms are calculated by the Image EP program”
After each trial, all arms and the center area are cleaned with super hypochlorous water to prevent bias based on olfactory cues
Note: Super hypochlorous water is an efficient odor removal agent with relatively weak odor compared to other cleaning solutions
“After each trial, all arms and the center area are cleaned with super hypochlorous water, that is an efficient odor removal agent and has relatively weak odor of itself compared to other cleaning solutions, to prevent a bias based on olfactory cues”
The order of trials is counterbalanced across genotypes because trial number affects the time spent in the open arms and center platform
Note: Indices increase during 3rd and 4th mice compared to 1st mice; counterbalancing controls for this effect
“The order of trials is counterbalanced across genotypes. That is, the indices all increase during the 3rd and 4th mice compared to the 1st mice”
Behavioral testing is performed between 9:00 AM and 6:00 PM
Note: Standardized time window for all testing
“Behavioral testing is performed between 9:00 AM and 6:00 PM”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Measure anxiety-like behavior in mice based on their aversion to open and elevated areas and natural exploratory behavior
Objective
Measure anxiety-like behavior in mice based on their aversion to open and elevated areas and natural exploratory behavior
Subjects
From papermouse • Not specified in protocol; C57BL/6J used for reference data • Not specified • Not specified • Not specified
Sample count
From paperNot specified per trial; reference data from n=914 C57BL/6J mice
Cohort notes
From paperMice housed with 12-h light/dark cycle (lights on at 7:00 AM); transferred to behavior testing room 30 min prior to testing
Animal housing and light cycle (Continuous)
Pre-testing habituation (30 minutes)
Practice trial with test animal (Not specified)
Mouse placement in maze (Immediate)
Number of entries into open arms
From paperThe Image EP program automatically calculates distance traveled, number of entries into each arm, time spent in each arm, and percent of entries into the open arms.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Number of entries into closed arms
From paperThe Image EP program automatically calculates distance traveled, number of entries into each arm, time spent in each arm, and percent of entries into the open arms.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Time spent in open arms (seconds)
From paperThe Image EP program automatically calculates distance traveled, number of entries into each arm, time spent in each arm, and percent of entries into the open arms.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Time spent in closed arms (seconds)
From paperThe Image EP program automatically calculates distance traveled, number of entries into each arm, time spent in each arm, and percent of entries into the open arms.
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Number of entries into open arms
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Number of entries into closed arms
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Time spent in open arms (seconds)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Time spent in closed arms (seconds)
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
The Image EP program automatically calculates distance traveled, number of entries into each arm, time spent in each arm, and percent of entries into the open arms.
Scoring or quantification
Quantify the primary readouts for this experiment: Number of entries into open arms; Number of entries into closed arms; Time spent in open arms (seconds); Time spent in closed arms (seconds).
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Number of entries into open arms, Number of entries into closed arms, Time spent in open arms (seconds), Time spent in closed arms (seconds).
Source links and direct wording from the methods section for validation and deeper review.
Citation
Munekazu Komada et al. (2008). Elevated Plus Maze for Mice. Journal of Visualized Experiments
Animal housing and light cycle • Protocol step
“Mice are housed with a 12-h light/dark cycle (lights on at 7:00 AM), as previously described”
Pre-testing habituation • Protocol step
“All the experimental mice are transferred to the behavior testing room 30 min prior to beginning the first trial to habituate to the condition of the behavior testing room”
Practice trial with test animal • Protocol step
“There are two purposes of the test trial using a practice animal. One is to make sure that everything is fine with the recording system. Another is to keep the testing condition as uniform as possible”
Mouse placement in maze • Protocol step
“A mouse is placed in the center area of the maze with its head directed toward a closed arm”
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Ohara & Co. • Not specified • Not specified • Not mentioned
Ohara & Co. • Not specified • Not specified • Not mentioned
Ohara & Co. • Not specified • Not specified • Not mentioned
Not specified • Not specified • Not specified • Not mentioned
Not specified • Not specified • Not specified • Not mentioned
O'Hara & Co., Tokyo, Japan (modified by Tsuyoshi Miyakawa) • Not mentioned
National Institute of Mental Health (developed by Wayne Rasband) • Not mentioned
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Steps
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Evidence Quotes
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Protocol Items
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Linked Products
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Evidence
9
Specific Products
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Canonical Sync
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