The Zero is an elevated ring-shaped runway with the same amount of area devoted to adjacent open and closed quadrants, with increasing usage in recent years. It is very similar to the elevated plus maze, but with the center region of the elevated plus maze removed. It has been pharmacologically validated with various anxiolytic drugs.
The design is created with an elevated circular platform with two walled, enclosed portions as well as two open portions, allowing for clear interpretations of murine behavior. Because mice tend to avoid open spaced areas, the animals will preferentially spend more time in the enclosed walled area.
$ 1890+ Shipping and Handling (approx $200)
- No Odors
- Matted for non reflective surfaces
- Easy clean with 70% Ethanol
$ 2090+ Shipping and Handling (approx $300)
- No Odors
- Matted for non reflective surfaces
- Easy clean with 70% Ethanol
The elevated zero maze is a variation of the elevated plus maze and is used to study anxiety-like behaviors in rodents. The behavioral task is based on the fact that rodents will generally avoid being exposed in the open and prefer enclosed areas. The maze is in the shape of a circle, with alternating enclosed and open quadrants. Information regarding the anxiety state of an animal can be gained by observing the amount of time spent in the closed or open areas of the maze. This maze is considered an improvement on the plus maze because it lacks a central platform, allowing it to be a more sensitive tool for anxiety behaviors and prevents confusing interpretations caused by the animal’s decision making. The circular shape of the zero maze allows animals to continually explore and forces them to choose between an open or enclosed area. This maze has been validated for successful use with anxiolytic drugs, as treated animals are more willing to explore the open areas of the maze than untreated animals.
The elevated plus maze was first designed based on a series of experiments and observations performed by K. C. Montgomery in 1955. Several decades after its initial use, it was validated for both rats and mice and proven sensitive to both anxiolytic and anxiogenic drugs in many studies in the late 1980s and early 1990s (Pellow et al. 1985, Lister 1987, Shepherd et al. 1994). One common issue among these studies was the interpretation of the time the animals spent on the central platform. To solve this problem, Jon K. Shepherd designed a similar maze that is now known as the zero maze (Shepherd 1992, Shepherd et al. 1993). This maze was in the shape of a circle with alternating closed and open quadrants, with no central platform. Since its initial introduction, the zero maze has proven just as successful in the study of anxiolytic and anxiogenic drugs as the plus maze (Shepherd et al. 1994).
The apparatus used for the zero maze consists of a raised circular alley divided into quadrants. The diameter of the maze can range from 40 to 110 cm, and the width of the alley can range from 5 to 10 cm, depending on whether mice or rats will be tested. Opposite quadrants are enclosed with dark-colored walls that are approximately 30 cm high, while the other two remain open and bordered with a low wall to prevent falls.
The light within all quadrants of the maze should be as equal as possible. This can best be achieved using an over-head light source.
A mounted video camera is used to record the experiments from above the maze. Tracking software can be used to follow the moments of the animals.
The purpose of the zero maze is to evaluate anxiety-like behaviors in mice and rat models by observing their willingness to explore the open areas of the maze. Other behaviors, such as head dips and rearing, also provide information regarding the animals’ anxiety state. These behaviors can be compared across control and intervention groups to see the effect of anxiolytic or anxiogenic drugs (Shepherd et al. 1994, Braun et al. 2010, Heredia et al. 2013). Typically animals with less anxiety will show shorter latency times, which is the time it takes the animal to enter an open area of the maze, and a higher percentage of time in the open areas.
For many tasks designed for rodents, the animals become accustomed to the maze and task over repeated exposures and trials. However, for the zero maze, several studies have shown that repeated trials actually increase anxiety in the animals (Treit et al. 1993, Cook et al. 2002). These studies suggest that the emotional response to the maze and task change with repeated testing. This agrees with what was shown for the elevated plus maze, where it was shown that repeated trials increased open arm avoidance in many animals (Treit et al. 1993). This tendency is important to remember when planning zero maze experiments and analyzing experimental results.
Evaluation of Anxiety-like Behaviors in the Zero Maze
This test does not require any prior training of the animals to be tested. The tendency for rodents to prefer dark, enclosed spaces and their willingness to explore are natural behaviors. Observing these behaviors in the maze will allow changes in these tendencies caused by treatment of drugs that affect anxiety to be seen.
To conduct the test, clean and prepare the maze apparatus. Bring animals into the room, and allow and an acclimation period if necessary. Start the video recording before placing animals into the maze.
Generally, a five-minute testing period is used. Carefully place the first animal into the center of one of the enclosed quadrants of the maze. Start a timer. Allow the animal to explore and move freely within the maze for five minutes. At the end of the testing period, carefully remove the animal from the maze and return to its home cage. Stop the recording. Repeat this trial for each animal on each testing day. Generally, animals are tested on three consecutive days.
Since Shepard initially designed the zero maze, few modifications have been made to its design. Most of the modifications published in studies utilizing the zero maze have been the use of different animal strains, many of which exhibit significantly different behaviors in the maze, or differences in the statistical analysis.
Several measures of data are collected from the zero maze. These include the latency time to enter an open quadrant, the total time spent in open and closed quadrants, the percentage of time spent in open and closed quadrants, and behaviors within open and closed quadrants. The video recordings of each trial are scored and analyzed using a stopwatch to determine the time spent by the animal in each quadrant. Other behaviors, such as grooming and head dips, should also be timed and tallied. This data is generally visualized by graphing the individual measures and then comparing across control and intervention groups of animals. If the animals are treated with anxiety-reducing drugs, the time spent in the open areas will be greater than the control animals, as shown in the example graph below. Behaviors associated with reduced anxiety, such as head dips and stretch attend postures, will be increased in animals treated with these drugs, also as shown below.
Using graphs similar to these will allow for easy comparison among animals treated with anxiety-reducing drugs and the sham control group. Animals treated with saline are naturally cautious of open, exposed areas and will have higher latency times, as well as a decreased percentage of time spent in open areas. Those animals treated with anxiolytic drugs, such as diazepam and chlordiazepoxide used as examples above, will show decreased latencies and a higher percentage of time spent in open areas. These animals will also likely display fewer anxiety-related behaviors, such as stretched attend postures. Generally, animal cohorts of approximately 10 animals are sufficient to obtain p-values of <0.05 using a 1-way ANOVA, ANCOVA, or post hoc tests (Shepherd et al. 1994, Cook et al. 2002, Braun et al. 2010, Heredia et al. 2013).
A major strength of the zero maze is its simplicity, both in terms of testing method and interpretation. The zero is regarded as an improvement on the older elevated plus maze because it does not contain a central platform. When analyzing rodent behavior in the plus maze, it became difficult to evaluate the time the animals spent on the central platform, either prior to entering an arm or between arm entries. The zero maze eliminates this central area and allows animals to continuously explore the different quadrants of the maze by moving between open and closed areas. This removes any ambiguity regarding the time spent on the central platform that is associated with the elevated plus maze (Shepherd 1994).
It is also important to remember that the zero maze examines emotions in animals that are comparative to feelings of anxiety in humans. However, this test is not an equivalent of the entire spectrum of human anxiety. It is evident that anxiolytic drugs are able to reduce anxiety-like behaviors in the animals in the maze, but there are other factors to consider that may play important roles in interpreting results, such as age, weight, sex, species, and strains of the animals tested.
Braun, A.A., Skelton, M.R., Vorhees, C.V., Williams, M.T. Comparison of the elevated plus and elevated zero mazes in treated and untreated male Sprague-Dawley rats: Effects of anxiolytic and anxiogenic agents. Pharm. Biochem. Behav. 97, 406-415 (2011).
Heredia, L., Torrente, M., Colomina, M.T., Domingo, J.L. Assessing anxiety in C57BL/6J mice: A pharmacological characterization of the zero maze test. J. Pharm. Tox. Methods 68, 275-283 (2013).
Montgomery, K.C. The relation between fear induced by novel stimulation and exploratory behaviour. J. Comp. Physiol. Psychol. 48, 254-260 (1955).
Shepherd, J.K. Preliminary evaluation of an elevated “zero-maze” as a model of anxiety in laboratory rats. J. Psychopharmacology 6, 223 (1992).
Shepherd, J.K., Grewal, S.S., Fletcher, A., Bill, D.J., Dourish, C.T. Behavioral and pharmacological characterization of the elevated “zero-maze” as an animal model of anxiety. Psychopharmacology 116, 56-64 (1994).