As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
View Abstract
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.
Protocol Steps
1
Animal housing and light cycle
Mice are housed with a 12-hour light/dark cycle with lights on at 7:00 AM
ContinuousNot specified
Note: Standard housing conditions maintained throughout study
View evidence from paper
“Mice are housed with a 12-h light/dark cycle (lights on at 7:00 AM), as previously described”
2
Pre-testing habituation
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
30 minutesNot specified
Note: Allows mice to acclimate to testing environment
View evidence from paper
“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”
3
Practice trial with test animal
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
Not specifiedNot specified
Note: The first mouse in entire session would experience different conditions without this practice trial
View evidence from paper
“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”
4
Mouse placement in maze
A mouse is placed in the center area of the maze with its head directed toward a closed arm
ImmediateNot specified
Note: Standardized starting position and orientation
View evidence from paper
“A mouse is placed in the center area of the maze with its head directed toward a closed arm”
5
Free exploration and recording
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
View evidence from paper
“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”
6
Data acquisition and analysis
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
Automated during recordingNot specified
Note: Image EP is based on public domain Image J program modified by Tsuyoshi Miyakawa
View evidence from paper
“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”
7
Maze cleaning between trials
After each trial, all arms and the center area are cleaned with super hypochlorous water to prevent bias based on olfactory cues
Not specifiedNot specified
Note: Super hypochlorous water is an efficient odor removal agent with relatively weak odor compared to other cleaning solutions
View evidence from paper
“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”
8
Trial order counterbalancing
The order of trials is counterbalanced across genotypes because trial number affects the time spent in the open arms and center platform
Not specifiedNot specified
Note: Indices increase during 3rd and 4th mice compared to 1st mice; counterbalancing controls for this effect
View evidence from paper
“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”
9
Testing time window
Behavioral testing is performed between 9:00 AM and 6:00 PM
9:00 AM to 6:00 PMNot specified
Note: Standardized time window for all testing
View evidence from paper
“Behavioral testing is performed between 9:00 AM and 6:00 PM”
Subjects / Specimens
Species
mouse
Strain
Not specified in protocol; C57BL/6J used for reference data
Age
Not specified
Sex
Not specified
Count
Not specified per trial; reference data from n=914 C57BL/6J mice
Weight
Not specified
Mice housed with 12-h light/dark cycle (lights on at 7:00 AM); transferred to behavior testing room 30 min prior to testing
