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Complex Behaviors

Overview: Anxiety and Depression Assays

By March 2, 2021No Comments

1. Introduction

Anxiety is characterized by prolonged apprehensive feelings to a threat that may not necessarily exist or be considered a threat. Classification of anxiety disorders is based on the symptoms experienced and their specific features. Sigmund Freud’s concept of anxiety suggests that it is composed of cognitive, affective, and behavioral components (Freud, 1936). Individuals with anxiety and anxiety disorders, in addition to the maladaptive flight or fight response, also experience psychophysiological symptoms such as sweating, disrupted sleep, and nausea. A combination of different factors, such as neurological changes, genetics, and environmental stress, potentially contribute to this emotional processing dysfunction.

Anxiety disorders range from panic disorders to phobias to stress-related disorders. More often than not, anxiety results in depression or depression contributes to the development of an anxiety disorder. The comorbidity of these two mental illnesses heavily impacts the overall quality of life; understanding of how these illnesses are experienced and processed is thus vital. Since individualistic differences make the assessment of these illnesses difficult, animal models serve as an important instrument in the development of drug treatments and understanding the neural basis of these disorders. However, the animal-based investigation may not be able to replicate all the features within a single model.

2. Animal Anxiety and Depression Assays

Animal anxiety assays often utilize the animal’s innate fears, such as falling or drowning in the evaluation of the underlying neural mechanisms and behavioral changes. Depression related investigations are usually geared toward assessing social interactions or reward-seeking behaviors.

The use of an automated tracking and recording system such as the Noldus EthoVision XT to assist with the observations is recommended in anxiety and depression assays.

2.1 Light-Dark Assays

The light-dark assays involve choice-based apparatuses wherein the subject has to choose between an enclosed, dark space or an open, illuminated open space. A reduction in anxiety behavior in these assays is measured by the time spent by the subjects in the open areas. The distance covered and the number of visits to the open, illuminated area also serves as a measure of anxiety.

Light-Dark Box

The Light-Dark Box is a two-chambered apparatus that consists of a dark enclosed space and an open illuminate area connected by a small door. The apparatus is also available as a Circular Light-Dark Box and Zebrafish Light-Dark Tank.

Elevated Plus Maze

The Elevated Plus Maze combines two aversive motivations; fear of falling and open, bright space. The maze is raised above the floor and consists of a central area from which four choice-arms extend. The choice arms are all perpendicular to the central area, with two opposite arms having high walls along its perimeter while the remaining two are unwalled. The walled arms serve as a safe space. A variation of the Elevated Plus Maze is the Asymmetric Elevated Plus Maze. The Asymmetric Elevated Plus Maze uses a combination of wall and no-wall set-up for each arm, creating a more anxiogenic environment for testing.

Elevated Zero Maze

The Elevated Zero Maze is an elevated continuous track unlike the Elevated Plus Maze and was developed by Shepherd in 1992. The track is divided into quadrants with two opposite quadrants having high walls while the remaining track is unwalled. The continuous track eliminates the ambiguity in choice behavior observation often associated with the Elevated Plus Maze.

I Maze

The I Maze apparatus consists of a single, elevated straight alleyway. The alleyway is divided into two parts, a closed start area that is equipped with a water spout and a configurable test arm. The two parts of the alleyway can be separated by a guillotine door. The configurable arm allows the option to either create an open arm or an enclosed arm similar to the start area. The apparatus set-up allows the evaluation of anxiety-related explorative behaviors.

2.2 Elevation-Based Assays

Elevation-based assays incorporate fear of falling as the primary motivation during the task. The subjects in these assays are subjected to the exploration of elevated, open spaces, often without any safe space option. A decrease in anxiety-related behavior is marked by an increased willingness to explore and spent time in the elevated, open area.

Successive Alleys

The Successive Alleys apparatus is a benchtop apparatus that consists of an enclosed space with a nested alleyway. The nested alleyway consists of four (including the start alley) sequentially narrowing alleys varying from dark to light color. The extended alleyway is designated as the open end, which hangs over the floor. The colors of the alleyway are in the order black, dark gray, light gray, and white, playing into the rodent’s preference of darker spaces.

3D Radial Arm Maze

The 3D Radial Arm Maze is a take on the traditional Radial Arm Maze. The apparatus consists of an elevated Radial Arm Maze with flexible arms that can be configured to be flattened, raised, and lowered. Additionally, the apparatus provides the opportunity to place cue plates at the ends of the choice arms, which allows for the application of different protocols. The apparatus was developed by Ennaceur, Michalikova, van Rensburg, and Chazot (2008).

Ladder Rewarded Elevated Plus Maze

The apparatus was first described by Bettis and Jacobs (2009) and is an adaptation of the conventional Elevated Plus Maze. Unlike the Elevated Plus Maze, the Ladder Rewarded Elevated Plus Maze does not include any walled arms. Additionally, each arm is equipped with a mesh bump that the subject must climb over in order to explore the arms. The subject is motivated by the availability of a ladder that is hidden at the end of the correct arm.

Height Test

The Height Test assesses anxiety-related behaviors by subjecting the animal to jump between platforms. The apparatus consists of a chamber with a raised unstable platform and two stable platforms placed at two distance under the unstable platform. One of the stable platforms is placed 2 cm under the unstable one, while the second stable platform is 50 cm below. The close-by platform appears safer than the further-away stable platform. Thus, the subject’s choice between these stable platforms allows assessment of their risk-taking capacity in terms of anxiety-related behaviors. The test shares some similarities to the Visual Cliff Test.

Elevated T-Maze

The Elevated T-Maze is an adaptation of the conventional T-Maze and was developed by Graeff, Viana, and Tomaz (1993). The maze consists of an enclosed arm at the end of which two open arms extend perpendicularly on either side. The test involves placing the subject at the distal end of one of the open arms or within the enclosed arm and observing the time it takes to explore the other arms.

2.3 Exploration Assays

Exploration assays are usually performed in open arenas using rodent’s innate fear of brightly lit open spaces. These assays may or may not involve an element of learning and/or escape spaces.


The Open-Field arenas are usually rectangular or circular and allow assessment of anxiety-related exploratory behaviors. The animals with a decreased level of anxiety are more likely to explore the central area of the open arena than animals with high levels of anxiety. The animals with high levels of anxiety will display reduced locomotion and exploration, with a preference to stay close to the walls of the open-field arena.

Barnes Maze

The Barnes Maze is an elevated, open circular platform with escape holes along the edge of the perimeter. One of the escape holes is equipped with a safe refuge, while the others serve as false escapes.

Escape Hole Radial Arm Maze

The Escape Hole Radial Arm Maze is an adaptation of the Radial Arm Maze. The maze is elevated above the floor and consists of a large, central open arena with eight (unwalled) arms radiating outwards. The ends of the arms are equipped with escape holes, usually, one serving as the true escape.

Morris Water Maze

The Morris Water Maze consists of an open, pool of water from which the subjects are expected to escape using the hidden escape platforms. In addition to the aversive motivation of open spaces, the Morris Water Maze also utilizes the fear of drowning. The assay is used in evaluating stress-related behaviors in learning and memory.

2.4 Miscellaneous Assays

Triple Test Maze

The apparatus sequentially combines a square Open-Field, an Elevated Plus Maze, and a Light-Dark Box to allow all in one evaluation of anxiety and depression-related behaviors. The apparatus was developed by Ramos, Pereira, Martins, Wehrmeister, and Izidio (2008) to allow single-trial evaluations.

Tail Suspension

The Tail Suspension apparatus includes compartments for multiple animal testing. The test involves suspending rodents by their tails and observing time spent immobile. The test is commonly used for drug testing.

Forced Swim Test

The Forced Swim apparatus includes a single tank with no escape. The test involves placing the rodents in the water and observing the time spent immobile. The test is commonly used for drug testing.

Chronic Social Defeat Stress Cage

The apparatus consists of a chamber divided by a removable, perforated transparent wall. The apparatus is used in the application of the resident-intruder paradigm similar to the Resident-Intruder Chamber task to evaluate the effects of social defeat on anxiety-related behaviors.

Fear Conditioning Chamber

The Fear Conditioning Chamber is used in the classical conditioning paradigm to evaluate fear behaviors as part of the analysis of anxiety and anxiety disorders. The apparatus consists of an animal holding chamber that is equipped with an electrified grid, light cues, and speaker. The entire chamber is placed with a sound attenuated isolation chamber.

Step-down Avoidance Chamber

The apparatus consists of a chamber equipped with an electrified grid flooring with a slightly elevated, vibrating circular platform placed at its center. The task was developed by Hudson B. B. in the 1950s as a one-trial Inhibitory Avoidance Task.

3. Human Anxiety and Depression Assays

In the clinical set-up, anxiety and depression are often assessed based on self-report and other diagnostic questionnaires (For digital healthcare tools visit Qolty). Empirical investigations through surveys, observational research, and correlational methods also provide insights into nature and factors that influence anxiety and depression processes. While these do provide relevant insights; emotions, and social understandings of humans are subjective in nature. Additionally, the investigator’s own bias may also influence the data apart from other experimental factors that cannot be controlled and kept consistent.

With the advancement of technology, more researchers are opting for virtual reality-based investigations that eliminate few of the ethical concerns associated with human-based research (For virtual reality tools visit Simian Labs). Virtual reality environments offer researchers great control over the simulation and the variables of task interactions. This then allows the creation of experiments that are more specific and controlled across all participants. Human virtual reality experiments also allow simulation of difficult scenarios and dangerous situations, such as death-related anxiety scenarios and traumatic experiences. For human virtual reality experiments, click here.

4. Ethical Obligations and Considerations

Animal models provide great assistance when it comes to drug testing and the development of treatments for anxiety and depression associated with many neuropsychiatric disorders and diseases. It is important that efforts be made to perform all investigations as ethically as possible. The following are few guidelines for animal-based experiments,

  • Social isolation for some animals can be extremely stressful and thus should be kept to the required minimum for an experiment.
  • Animals should be habituated to handling to minimize the effects of handling stress.
  • Experiments that involve aggressive interactions should be carefully monitored to prevent injury to the animals involved.
  • Animals should be habituated to restraint to prevent unnecessary fear and stress. (For restraints click here).
  • Apparatuses should be cleaned as necessary to prevent any lingering olfactory cue from influencing the subject behavior.

Apart from the above guidelines, efforts should be made to ensure the overall wellbeing of the animals in the laboratory. Animals should not be subjected to unnecessary stress or mishandling at any time.

In human experiments and research, the following are few guidelines that should be followed,

  • Explicit consent of the participants should be obtained prior to testing.
  • Experiments should be age-appropriate, and all medical factors should be taken into consideration.
  • Safety and well-being of the participants should be prioritized above all.
  • Experiments that involve potential triggering set-ups should be carefully created so as not to overwhelm or stress the participant.
  • Appropriate measures should be taken when using virtual reality for experimentation.

5. Conclusion

Animal models allow the development of treatments and drugs to ameliorate the debilitating symptoms of anxiety and depression disorders. These investigations shed light on the neural mechanisms involved, possible genetic factors, and environmental factors that contribute to the development of these disorders. While an irreplaceable tool in mental illness research, animal models cannot replicate all the symptoms and features of a human disorder. Further, experiences that are relevant only to humans may not necessarily be accurately modeled in animals. Thus, translating the findings of animal models to humans should be done with great consideration.


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  2. Bettis, T.J., & Jacobs, L.F. (2009). Sex-specific strategies in spatial orientation in C57BL/6J mice. Behavioural Processes; 82(3):249-55. doi: 10.1016/j.beproc.2009.07.004.
  3. Ennaceur, A., Michalikova, S., van Rensburg, R., & Chazot, P.L. (2008). Are benzodiazepines really anxiolytic? Evidence from a 3D maze spatial navigation task. Behavioural Brain Research; 188(1):136-53. DOI: 10.1016/j.bbr.2007.10.026
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  6. Hudson B. B. (1950). One-trial learning in the domestic rat. Genetic Psychology Monographs; 41(First Half):99-145
  7. Ramos, A., Pereira, E., Martins, G.C., Wehrmeister, T.D., & Izídio, G.S. (2008). Integrating the open field, elevated plus maze and light/dark box to assess different types of emotional behaviors in one single trial. Behavioural Brain Research; 193(2):277-88. doi: 10.1016/j.bbr.2008.06.007.
  8. Shepherd, J.K. (1992). Preliminary evaluation of an elevated “zero-maze” as a model of anxiety in laboratory rats. Journal of Psychopharmacology, 6, 223
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