Zero Maze

Elevated Zero Maze (EZM) is an adaptation of the Elevated Plus Maze (EPM) that combines both classical and new ethological measures. The maze has been gaining steady popularity as a behavioral assay for anxiety-like behaviors. It takes advantage of a rodent’s innate preference for dark, enclosed spaces as opposed to open spaces. Unlike the EPM, the EZM subjects the participant to a continuous circuit that has alternating walled and un-walled areas.

The maze was developed based on the principles of Montgomery (1955) experiment that used an elevated Y-Maze with one of the choice arm enclosed to mimic a safe space and the other choice arm left un-walled. The Elevated Zero Maze was developed (Shepherd 1992, Shepherd et al.,1993) and pharmacologically validated for anxiety-like behaviors in mice by Shepherd and colleagues in their paper published in 1994. The maze has since seen a growing interest as a better alternative to the Elevated Plus Maze.

The Elevated Zero Maze consists of a circular track that is elevated above the ground and divided into four quadrants. Alternating segments (quadrants) of the alleyway are enclosed by dark walls while the rest remain open. The EZM eliminates a choice point as seen with the EPM thereby removing the ambiguity associated with a central arena.

Origin

The Elevated Zero Maze was first described by Shepherd (Shepherd 1992). It was designed to overcome the issue of the Elevated Plus Maze. The EPM’s central platform, which served as the choice point for the subject to choose one of the four arms, resulted in difficulty in interpretation of the time spent in that area. To overcome this ambiguity presented by the EPM, Shepherd designed a circular maze with alternating enclosed and open areas.

The pharmacological validation of the Elevated Zero Maze was done by Shepherd and colleagues in the preceding papers (Shepherd et al.,1993, Shepherd et al.,1994). The apparatus showed great success as an animal model of anxiety and proved to be a more sensitive test in comparison to the EPM.

Developments

Matto et al. used the EZM to evaluate the effects of cholecystokinin A and B receptor agonists SR 27897 B, devazepide, L 365260 and PD 135158 (CAM 1028) on exploratory behaviors of rats. Their experiment was able to support the involvement of CCK(B) receptor subtype in the neurobiological mechanisms of anxiety.

The differential effects of beta-carbolines and antidepressant were explored by Pähkla et al.  In their paper, they compared the antidepressants desipramine and fluoxetine with anxiogenic beta-carboline DMCM. They further tested pinoline in comparison to DMCM and the antidepressants. The results of their investigation suggested that in comparison to the antidepressants both pinoline and DMCM decreased the time spent in open spaces, the number of line crossings in the open spaces and the number of head dips. They were able to conclude that mechanisms other than serotoninergic in nature were involved in the action of pinoline.

mGlu1 receptor blockade’s effect on anxiety-like behaviors in rats was tested by Steckler et al. Doses of up to 10mg/kg IP of JNJ16259685 showed to have no anxiolytic effect on the performance of the rats on the Elevated Zero Maze. However, on treatment with lowest active dose (5mg/kg) of benzodiazepine chlordiazepoxide a relative increase in time spent in open spaces was observed.

Recent Developments

Using mice deficient in either mGlu4(-/-) or mGlu8(-/-) or both receptors mGlu4/8(-/-) the possible relation and overlapping of functions of the mGlu4 and mGlu8 metabotropic receptors was analyzed by Davis et al. When subjected to the Elevated Zero Maze, mGlu4 deficient mice spent more time in open areas of the maze in comparison to the other groups. However, no genotype difference in activity levels was observed for any of the groups.

The anxiety-like behaviors of APP/PS1 mice were tested using the EZM in their investigation by Biallosterski et al. The mice were subjected to the EZM after voiding behavior testing, and it was observed that the APP/PS1 mice showed a lower preference for open space as compared to the wild-type mice that spent significantly more time in the open sectors.

Tucker et al. tested C57BL/6J mice in the EZM after controlled cortical impact (CCI) to investigate the anxiety-like symptoms following a traumatic brain injury. In their investigation, they observed that CCI reduced anxiety-like behaviors and fewer anxiety-like behaviors were associated with the female sex.

The Zero Maze is composed of a circular alley of diameter ranging from 40 to 110 cm and having a width in the range 50 to 10 cm. The alleyway is divided into four equal quadrants. Each of the opposite quadrants is walled with approximately 30 cm high, dark-colored walls to create an enclosed space. The remaining two quadrants are bordered with an approximately 5mm rim in order to prevent the subject from falling off the track, and these quadrants serve as the open anxiogenic spaces. The apparatus is raised above the floor to a height of at least 70 cm.

To avoid shadows and to ensure equal lighting in all the quadrants, the EZM should be lit from above. Proper lighting also ensures that the subject is able to see the food rewards or other cues if used.  A camera connected to tracking software such as ConductVision, Noldus Ethovision XT,and ANY-Maze can mounted above the maze to assist with live scoring, tracking and recording the subject and its movements within the maze. The apparatus must be cleaned thoroughly before and after each trial to limit influence from any residual stimuli from previous trials.

The Elevated Zero Maze’s continuous alley design serves as a more sensitive maze than the Elevated Plus Maze and thus is employed in the assessment of anxiety and anxiety-like behaviors. Like the EPM, the EZM also utilizes the subject’s preference for open spaces as a way to assess the performance of the treated group or the disease group in comparison to a control group.

Rodents have an innate preference for closed or enclosed spaces where they feel safe. Thus they tend to avoid exploring exposed alleys as much as the enclosed ones. Animals treated with anxiolytic drugs show reduced anxiety-like behavior and are more likely to explore the exposed areas of the maze. On the other hand, animals treated with anxiogenic drugs tend to spend less time in open spaces and more time exploring the enclosed areas due to the heightened anxiety-like feeling. The amount of time the animal spends on either of the areas provides information regarding the anxious state of the subject.

The maze as such requires no pre-training as the task is intended to test the response of the subject to an unfamiliar maze. Before every trial, the apparatus must be thoroughly cleaned with a solution of ethanol to avoid the influence of residual stimuli if any.

Evaluation of Anxiety-like Behaviors in the Zero Maze

The subjects are brought into the testing area at least 1 hour prior to the testing. During the sessions, it is important to ensure all the lighting level are consistent and do not inhibit exploratory behaviors.

The video recording is started (if being used) and the subject is placed in the center of the enclosed alley and allowed to freely explore the maze (Some investigations place the subject in the center of the open area). Each testing session lasts for at least 5 minutes after which the subject is carefully removed from the maze and placed in its home cage. The recording is also stopped at the expiration of the 5 minutes.

Trials are repeated for each subject for over a period of about 3 days.

The Elevated Zero Maze has seen minimal variation in its general apparatus design since Shepherd’s initial design; however, it has been adapted for different laboratory animals. Although design modifications are yet to be seen, simple modifications such as using transparent walls and changing the length or frequency of the enclosed arenas could have potential in investigatory processes involving the EZM.

Most of the published modifications utilizing EZM have been with the use of different strains of rodents. Different strains of mice tend to exhibit significantly different behaviors in the maze, or differences in the statistical analysis. (Biallosterski et al.,2015; Tucker et al.,2017)

The EZM has also been frequently used in determining the role of gene modifications and analyzing effects of pharmacological substances. (Heredia et al.,2012; Wilking et al.,2012; Davis et al.,2013).

Similar to the Elevated Plus Maze, the variables measured during the Elevated Zero Maze task include

• Percentage time spent in the open areas
• Percentage time spent in the walled areas
• Latencies to enter the open spaces
• Total time spent in open and closed areas
• Total distance traveled

Other observable measures include,

• Risk assessment behavior calculated by analyzing the frequency and duration of sniffing from inside the closed area. (Schulz et al.,2011)
• Reduced level of anxiety by assessing the number of head dips over the edge of the open area. (Rodgers & Dalvi 1997)

General activity is determined as the number of entries into closed spaces. Subjects spending shorter intervals in the open spaces are translated as increased anxiety (Pellow et al.,1985). Behaviors associated with reduced anxiety, such as head dips and stretch attend postures, tend to increase in animals treated with anxiolytic drugs.

Using graphs 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 tend to 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 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).

The Elevated Zero Maze overcomes the difficulty that arises in Elevated Plus Maze in evaluating the biological significance of anxiety-related behavior due to the ambiguous central platform. Further, the EZM has a lower baseline level in comparison to the EPM thus making it more sensitive to changes (Braun et al.,2011).

Space voyages expose astronauts to an environment much different from that of the earth. Exposure to microgravity and ionizing radiations during lengthy voyages could have potential effects on physical abilities, mood, and cognitive functioning. An investigation into the possible effects of combined microgravity and low-dose radiation was conducted by Bellone et al.

Nobre MJ (2016) explored the potential of environment-enrichment in attenuating the adverse effects of withdrawal and social isolation on rat’s behavior. It has been observed that drug-addicted individuals show behavioral patterns similar to patients with lesions of the mPFC, which could potentially be mechanisms responsible for the transition from the casual to compulsive drug use.

Animal models of mood disorders often employ stressful environments as a comparative reflection of stressful lives of humans. Matrov et al. used a rat model of chronic social defeat to confirm that low levels of exploratory activity are a risk factor for passive stress coping. They employed EZM to observe the stress and explorative behaviors of the groups.

The therapeutic potential of direct cannabinoid agonists was investigated by Kathuria et al. In their paper, they suggested an alternative approach to developing drugs that amplify the effects of endogenous cannabinoids by preventing their inactivation.  They were able to demonstrate that URB532 (0.1–10 mg kg–1, i.p.) and URB597 (0.05–0.1 mg kg–1, i.p.) evoked anxiolytic-like responses at doses that corresponded to those required to inhibit FAAH activity in vivo in an EZM task.

The major strength of the Elevated Zero Maze is its simplicity, both in terms of testing method and interpretation. The EZM overcomes the ambiguity in measuring that arises from the EPM due to its central platform. Over repeated trials in the EPM, the subject tends to associate the central platform as a safe space. When analyzing rodent behavior in the EPM, it becomes difficult to evaluate the time the animals spent on the central platform, either prior to entering an arm or between arm entries. The EZM eliminates this central area and allows animals to continuously explore the different quadrants of the maze by moving between open and closed areas (Shepherd 1994).

Unlike other tasks, the subjects on repeated exposure to the Elevated Zero Maze show an increase in anxiety which suggests that the emotional responses of the subject change with every exposure to task on the maze. It is also important to remember that the EZM 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 can 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.

• The Elevated Zero Maze is an adaptation of the Elevated Plus Maze developed based on the principles of Montgomery (1955) investigation.
• The EZM was designed by Shepherd (1992).
• To overcome the ambiguity generated by the central platform in the EPM, the EZM was designed as a circular alley with closed and open spaces.
• The EZM is more sensitive in the assessment of anxiety-like behaviors than the EPM.
• Subjects treated with anxiolytic drugs tend to show lessened anxiety-like behavior by visiting the open area more.
• Subjects treated with anxiogenic drugs show heightened anxiety-like behavior and prefer the enclosed areas of the maze.
• Specific strains of rodents may perform differently on the maze.
• Repeated trials on the EZM tend to increase anxiety-like behavior.

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