Elevated Plus Maze for Screening Antianxiety Drugs

Introduction/Overview

The discovery and development of novel pharmacotherapies for anxiety disorders rely heavily on validated preclinical behavioral models. Among these, the Elevated Plus Maze (EPM) stands as one of the most widely employed and extensively characterized tests for the initial screening of potential anxiolytic compounds. Its utility stems from its ability to quantify approach-avoidance conflict, a core psychological construct in anxiety, by exploiting the natural aversion of rodents to open, elevated spaces while simultaneously appealing to their exploratory drive. The test provides a quantifiable behavioral output that is sensitive to established anxiolytic agents, making it a cornerstone in the preclinical pipeline for central nervous system drug discovery.

The clinical relevance of this model is paramount. Anxiety disorders, encompassing generalized anxiety disorder, panic disorder, social anxiety disorder, and specific phobias, represent a significant global burden of disease. The development of new therapeutic agents with improved efficacy, faster onset of action, and more favorable side-effect profiles compared to existing medications like benzodiazepines and selective serotonin reuptake inhibitors remains a critical goal. The EPM serves as a primary, high-throughput filter to identify compounds worthy of further investigation in more complex and costly models and, ultimately, clinical trials. Its predictive validity, while not absolute, has been instrumental in advancing our understanding of anxiolytic pharmacodynamics.

Learning Objectives

• Describe the fundamental principles, design, and standard procedural protocol of the Elevated Plus Maze test.
• Explain the behavioral rationale and theoretical underpinnings of the EPM as a model of anxiety-like behavior in rodents.
• Analyze the primary and secondary behavioral parameters measured in the EPM and their interpretation in the context of drug screening.
• Evaluate the predictive, face, and construct validity of the EPM for screening anxiolytic and anxiogenic compounds.
• Critically appraise the advantages, limitations, and crucial methodological variables that can influence EPM outcomes in pharmacological studies.

Principles and Design of the Elevated Plus Maze

The Elevated Plus Maze apparatus is a plus-shaped (+) platform elevated a significant distance from the floor, typically 50-70 cm for rats and 40-50 cm for mice. It consists of two open arms and two enclosed arms, each with high walls, arranged opposite each other. The open arms lack side walls, exposing the animal to the height and open space, while the enclosed arms provide a perceived safer environment. The maze is usually constructed from wood, plastic, or opaque acrylic, and testing is conducted under moderate, consistent illumination. The fundamental premise is the creation of an unconditioned conflict between the rodent’s innate exploratory behavior and its innate fear of open, elevated spaces. Anxiolytic drugs are expected to disinhibit behavior, increasing the proportion of time spent in and entries into the open arms, whereas anxiogenic manipulations should have the opposite effect.

Standard Testing Protocol

A standardized protocol is essential for reproducibility. Testing typically involves a single, brief session (usually 5 minutes) following a period of habituation to the testing room. The animal is placed in the central square of the maze, facing an open arm, and its behavior is recorded via an overhead video tracking system or by a trained observer blind to the treatment condition. Key parameters are quantified post-session. Prior handling of the animals for several days is recommended to minimize stress from experimenter interaction. The maze must be thoroughly cleaned between subjects with a mild disinfectant to eliminate olfactory cues that could influence the behavior of subsequent animals.

Behavioral Parameters and Pharmacological Interpretation

The behavioral output from an EPM session is reduced to several key metrics. The choice of parameters and their interpretation requires careful consideration.

Primary Measures

• Open Arm Time (OAT): The total time spent in the open arms. An increase in OAT is the most classic indicator of an anxiolytic effect.
• Open Arm Entries (OAE): The number of entries into the open arms. An entry is typically defined as all four paws crossing into the arm.
• Open Arm Time Percentage (%OAT): (Time in open arms ÷ Total time in all arms) × 100. This parameter controls for general locomotor activity.
• Open Arm Entry Percentage (%OAE): (Entries into open arms ÷ Total entries into all arms) × 100. Similarly, this ratio is less confounded by changes in overall exploration.

Secondary and Control Measures

• Total Arm Entries: The sum of entries into all four arms. This serves as a general index of locomotor activity. A significant decrease can indicate sedative or motor-impairing effects of a drug, which can confound the interpretation of reduced open arm exploration as an anxiogenic effect.
• Closed Arm Time: Time spent in the enclosed arms. Often inversely related to OAT.
• Risk Assessment Behaviors: More ethological measures, such as stretched-attend postures (SAP) where the animal stretches forward from a protected area to scan an open arm without entering, and head-dipping over the edges of the open arms. These are considered conflict behaviors and may be sensitive to different aspects of anxiolytic action.
• Rearing and Grooming: Can provide additional context on general activity and displacement behavior, respectively.

Pharmacological interpretation hinges on the pattern of changes. A prototypical anxiolytic (e.g., a benzodiazepine) increases %OAT and %OAE without reducing, or while possibly increasing, total arm entries. A compound that reduces open arm exploration while also drastically reducing total entries may be sedative rather than purely anxiogenic. Therefore, data should always be presented as both absolute values and percentages to allow for this critical distinction.

Pharmacological Validation and Predictive Validity

The EPM has been pharmacologically validated using a wide range of established anxiolytic and anxiogenic agents. Its predictive validity refers to its ability to correctly identify drugs with known clinical efficacy in treating human anxiety disorders.

Response to Standard Anxiolytic Agents

The maze is highly sensitive to several drug classes. Benzodiazepines (e.g., diazepam, chlordiazepoxide) produce a robust, dose-dependent increase in open arm exploration. 5-HT_1A receptor partial agonists (e.g., buspirone) also show anxiolytic profiles in the EPM, albeit sometimes with a different temporal profile or requiring specific testing conditions. Certain antidepressants with anxiolytic clinical effects, particularly selective serotonin reuptake inhibitors (SSRIs), can increase open arm activity, but their effects are often more variable and may require chronic, rather than acute, administration to manifest—mirroring their clinical latency. This underscores the importance of appropriate dosing regimens in screening.

Response to Anxiogenic Agents and Panicogens

The model also detects anxiogenic effects. Inverse agonists at the benzodiazepine site of the GABA_A receptor (e.g., FG-7142), noradrenergic stimulants like yohimbine (an α₂-adrenoceptor antagonist), and corticotropin-releasing factor (CRF) receptor agonists reliably decrease open arm exploration. This sensitivity enhances the utility of the EPM for studying the neurobiology of anxiety states and for screening compounds that may potentially induce anxiety as a side effect.

Limitations of Predictive Validity

It is crucial to recognize the limitations. Some clinically effective anxiolytics, such as certain tricyclic antidepressants, may not show robust effects in the standard EPM protocol. Furthermore, the test is primarily a model of generalized or innate anxiety rather than conditioned fear or panic. Drugs effective for specific anxiety subtypes may not be detected. False positives can occur; for example, psychostimulants like amphetamine can increase open arm entries, but this is likely due to general hyperactivity and disinhibition rather than a specific anxiolytic action, highlighting the need for control measures like total entries.

Neurobiological Substrates and Construct Validity

The behavioral output in the EPM is modulated by a complex neural circuitry, providing construct validity by linking the test to known neurobiological systems involved in human anxiety.

The aversive nature of the open arms is mediated by brain structures such as the amygdala, bed nucleus of the stria terminalis (BNST), and periaqueductal gray (PAG). GABAergic transmission, particularly via GABA_A receptors containing α₂, α₃, and α₅ subunits, plays a dominant inhibitory role, which is the target of benzodiazepines. Serotonergic pathways from the dorsal raphe nucleus to limbic structures exert a primarily anxiogenic influence, though this system is complex with multiple receptor subtypes (e.g., 5-HT_1A vs. 5-HT_2C) having opposing effects. Noradrenergic projections from the locus coeruleus and dopaminergic mesolimbic pathways also modulate conflict behavior. The test is sensitive to manipulations of the hypothalamic-pituitary-adrenal (HPA) axis, with corticosterone administration often being anxiogenic. This multi-system engagement makes the EPM a useful tool for probing the anxiolytic potential of compounds acting on diverse neurotransmitter targets.

Critical Methodological Variables and Best Practices

The outcome of an EPM experiment can be significantly influenced by numerous methodological factors. Standardization is key to reliable and reproducible drug screening.

Apparatus and Environmental Factors

• Arm Dimensions and Height: The relative width and length of open vs. closed arms, as well as the elevation of the maze, must be standardized. Inappropriate dimensions can lead to ceiling or floor effects in behavior.
• Illumination: Light intensity is a critical anxiogenic stimulus. Testing is usually done under moderate, diffuse light (e.g., 50-100 lux). High illumination increases baseline aversion to open arms, potentially masking anxiolytic effects.
• Room Characteristics: Extraneous visual cues, noise, and odors must be minimized and kept constant.

Animal-Related Factors

• Species and Strain: Mice and rats differ in baseline open arm exploration. Furthermore, significant inter-strain differences exist within species; some mouse strains (e.g., BALB/c) are highly anxious in the EPM, while others (e.g., C57BL/6) are more exploratory. Strain selection must be justified.
• Age and Sex: Age can affect performance, and there are well-documented sex differences in EPM behavior and pharmacological response, necessitating the inclusion of both males and females in modern screening paradigms where applicable.
• Prior Stress and Housing: Previous stress experiences (e.g., social defeat, restraint) and housing conditions (single vs. group) can alter baseline anxiety levels.

Experimental Design Factors

• Time of Testing: Rodents are nocturnal; testing during their active (dark) phase may yield different baseline behavior than during the light phase.
• Drug Administration Protocol: The route of administration (intraperitoneal, subcutaneous, oral), dose volume, vehicle used, and pre-test injection interval must be optimized for each compound based on its pharmacokinetic profile.
• Experimenter Effects: The presence of the experimenter in the room can be stressful. Remote video recording and automated tracking are preferred.

Integration in the Drug Discovery Pipeline

The EPM is rarely used in isolation. It typically occupies an early position in a hierarchical screening strategy.

A compound demonstrating a robust, dose-dependent anxiolytic effect in the EPM, without concomitant sedation or motor impairment, would be advanced to more complex and ethologically relevant models, such as the Vogel conflict test (for antidepressant/anxiolytic activity involving punishment) or social interaction test, to confirm its profile and rule out false positives.

Ethical Considerations and the 3Rs

The use of animals in anxiety research is governed by the principles of Replacement, Reduction, and Refinement (3Rs). The EPM is considered a moderate-stress procedure. Refinements include using the shortest possible test duration, providing highly controlled and consistent environments to minimize distress, and employing sensitive video-tracking to extract maximal data from each subject (Reduction). While in vitro models are developing, the complex, integrated nature of anxiety behavior currently necessitates the use of whole-animal models like the EPM. Justification of animal numbers through robust statistical power analysis is an ethical and scientific imperative.

Emerging Modifications and Technological Advances

Traditional EPM methodology continues to evolve. The integration of automated, high-definition video tracking software allows for the precise quantification not only of standard parameters but also of subtler ethological measures like gait, velocity, and complex risk-assessment sequences. Some researchers employ a “zero maze,” a circular elevated track with alternating open and closed sections, which eliminates the ambiguous central square. Furthermore, the EPM is being combined with modern neuroscientific techniques; for example, performing the test on animals with site-specific brain lesions, optogenetic or chemogenetic manipulations, or in vivo microdialysis to measure neurotransmitter release during the behavioral task. These approaches transform the EPM from a simple screening tool into a platform for dissecting the neural circuitry of anxiety.

Summary/Key Points

• The Elevated Plus Maze is a fundamental, unconditioned rodent model used for the primary behavioral screening of novel compounds for anxiolytic or anxiogenic potential.
• Its principle is based on the natural conflict between exploration and aversion to open, elevated spaces. Anxiolytic drugs typically increase the percentage of time spent in and entries into the open arms.
• Key outcome measures include open arm time percentage and open arm entry percentage, which must be interpreted alongside total arm entries to control for nonspecific effects on locomotor activity.
• The test demonstrates good predictive validity for several drug classes (e.g., benzodiazepines, 5-HT_1A agonists) but has limitations, including variable responses to chronic antidepressants and potential for false positives from stimulants.
• Behavior in the EPM is modulated by multiple neurobiological systems, including GABAergic, serotonergic, noradrenergic, and HPA axis pathways, reflecting the complexity of anxiety.
• Results are highly sensitive to methodological variables such as apparatus design, illumination, animal strain, sex, and testing protocol. Rigorous standardization is essential for reproducibility.
• In drug discovery, the EPM serves as a secondary screen. Positive hits require confirmation in additional, more specific behavioral models before progression to advanced preclinical development.

Clinical Pearls for Interpretation

• A compound that increases open arm exploration and total arm entries may possess both anxiolytic and stimulant properties, requiring careful follow-up.
• A reduction in open arm exploration coupled with a significant decrease in total entries is more indicative of sedation or motor impairment than a pure anxiogenic effect.
• The failure of a clinically effective drug (e.g., an SSRI) to show an effect in the standard acute EPM protocol does not invalidate the model but highlights the importance of aligning preclinical dosing regimens (e.g., chronic administration) with clinical use.
• When evaluating novel mechanisms, the EPM should be part of a battery of tests; activity across multiple models strengthens the case for a true anxiolytic profile.

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