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Elevated plus maze

The elevated plus maze (EPM) is a widely used behavioral assay in neuroscience to evaluate anxiety-related behaviors in rodents, such as mice and rats, by exploiting their innate aversion to open, elevated spaces and preference for enclosed areas. The apparatus consists of four arms arranged in a plus-shaped configuration—two opposite open arms without walls and two opposite enclosed arms with high walls—extending from a central platform and elevated approximately 40–50 cm above the ground, typically made of non-reflective materials to minimize visual cues. During a standard 5-minute trial, the rodent is placed in the central area and allowed to explore freely, with anxiety levels measured by the percentage of time spent in and entries into the open arms relative to the closed ones; reduced open-arm activity indicates higher anxiety.^[1]^[2] The EPM's development traces back to K.C. Montgomery's 1955 Y-maze, which examined exploratory behavior in rats by contrasting open and enclosed alleys to assess novelty-seeking and fear responses.^[3] This was adapted into the elevated plus configuration by S.L. Handley and S. Mithani in 1984 to enhance the conflict between exploration and aversion to height, providing a more sensitive model for unconditioned anxiety. The test was formally validated as a pharmacological screening tool for anxiolytic and anxiogenic agents by S. Pellow and colleagues in 1985, demonstrating its sensitivity to benzodiazepines and other compounds modulating GABAergic neurotransmission.^[2] Beyond anxiety assessment, the EPM has proven versatile in preclinical research, including evaluations of drug efficacy for psychiatric disorders, neurobiological mechanisms involving brain regions like the amygdala and hippocampus, and genetic or environmental influences on emotionality. It is particularly valued for its simplicity, requiring no prior training, and ethical advantages over more invasive methods, though variations such as lighting conditions or arm modifications are employed to address limitations like one-trial tolerance or strain-specific responses. Ongoing refinements continue to support its role in translational studies of anxiety and related conditions.^[1]^[2]

Introduction and History

Description

The elevated plus maze is a behavioral test primarily used to evaluate anxiety-like behaviors in rodents by exploiting their natural aversion to open, elevated spaces.^[4] This assay leverages the ethological principle that rodents innately prefer enclosed, dark environments over exposed, brightly lit ones, reflecting an adaptive fear response to potential predators and heights.^[5] The apparatus features a plus-shaped platform elevated above the ground, comprising two opposing open arms without walls and two opposing closed arms surrounded by walls, typically constructed from non-reflective materials such as matte black plastic or wood to minimize visual cues and glare.^[1] Standard dimensions generally include arms 30-50 cm long and 10-15 cm wide, with the entire structure raised 50-70 cm from the floor and closed arm walls 30-40 cm high.^[5] This design facilitates the observation of exploratory tendencies versus avoidance behaviors, serving as a tool for screening anxiolytic drugs and investigating anxiety-related mechanisms.

Development and Evolution

The roots of the elevated plus maze can be traced to foundational work on fear and exploratory behavior in rodents. In 1955, K.C. Montgomery developed an elevated Y-maze to study how novel stimulation induces fear while motivating exploration in rats, noting that animals preferentially avoided open arms due to perceived risk but entered enclosed arms more readily.^[6] The elevated plus maze itself emerged in 1984, when Sheila L. Handley and S. Mithani introduced the "elevated X-maze" as a novel apparatus for evaluating anxiolytic drug effects without relying on footshock-induced conflict, building directly on Montgomery's observations of open-arm aversion as a fear indicator.^[7] This design featured four arms in a cross configuration—two open and two walled—to exploit the natural conflict between exploration and aversion to height and openness, and it was initially tested for sensitivity to alpha-adrenoceptor agonists and antagonists in modulating fear-motivated behavior. Refinement and validation followed swiftly in 1985, with S. Pellow, S.E. File, and colleagues demonstrating in rats that entries and time spent in open versus closed arms provided a reliable, quantifiable measure of anxiety, responsive to established anxiolytics like chlordiazepoxide and insensitive to locomotor stimulants. The test was adapted for mice in 1987 by R.G. Lister, who established comparable protocols and confirmed its predictive validity for anxiolytic and anxiogenic compounds in this species, thereby facilitating cross-species comparisons and standardization.^[8] From the late 1980s onward, the elevated plus maze transitioned from a specialized anxiety screening tool to a cornerstone of neuroscience, with widespread adoption by the 1990s for probing neural circuits underlying emotional behavior.^[1] The 2000s saw deeper integration with pharmacology and genetics, as researchers employed it to evaluate drug mechanisms and genotype effects on anxiety-like responses in mutant rodent lines, amassing over 2,000 publications by mid-decade.^[1] By 2025, the number of publications exceeds 10,000, underscoring its enduring utility alongside ongoing refinements such as automated behavioral scoring and novel ethological measures.^[9] Among its milestones, the maze's validation against anti-anxiety agents in the 1980s cemented its pharmacological relevance, while expansions in the 2010s included virtual and mixed reality adaptations for human analogs, enhancing translational potential without altering the core ethological basis.^[10]

Apparatus and Methodology

Design Specifications

The elevated plus maze (EPM) apparatus is typically constructed from opaque materials such as black or dark gray plastic, wood, or metal to reduce reflections and prevent neophobia in rodents, with arms designed to minimize climbing by featuring smooth walls and ledges on open sections.^[1]^[11] The standard design for rats, originating from 1980s publications, includes two open arms measuring 35-50 cm in length and 10-12 cm in width without any enclosing walls, and two closed arms of identical length and width but surrounded by 30-40 cm high opaque walls, sometimes with an optional ledge or roof for added enclosure.^[7]^[11] A central platform, usually a 10 × 10 cm square, connects the four arms at their intersection to facilitate transitions.^[11]^[12] The entire apparatus is elevated 50-70 cm above the floor level to evoke mild height-induced anxiety without causing panic, ensuring the test remains sensitive to anxiolytic effects.^[11]^[12] Experiments are conducted in a controlled environment, such as a dimly lit room with illumination levels of 5-20 lux in the open arms to enhance the aversive nature of those areas, while maintaining low ambient noise to avoid confounding stressors.^[12]^[13] Between trials, the maze is cleaned with a mild detergent or 70% ethanol solution to eliminate olfactory cues from previous subjects.^[1] Optional features may include infrared sensors along arm edges for automated entry detection or integration with video tracking software to monitor movement precisely, as well as removable inserts for open arms to add shallow ledges (e.g., 0.5 cm high) that prevent falls while preserving the design's integrity.^[12]^[14]

Experimental Procedure

The experimental procedure for the elevated plus maze (EPM) test utilizes adult rodents, typically male or female rats weighing 200–300 g or mice weighing 20–30 g, to assess anxiety-like behavior without prior food or water deprivation unless specified for a particular study.^[4]^[15] Animals are gently transported to the testing area in covered carriers to minimize stress and disturbance, and they undergo habituation in the testing room for 30–60 minutes before the trial to acclimate to the environment and reduce novelty-induced anxiety.^[4]^[16] The procedure is designed as a single trial per animal to avoid learning effects or habituation to the apparatus.^[17]^[18] For trial execution, the rodent is placed in the center platform of the standard EPM apparatus—elevated approximately 50 cm above the floor—with its head facing an open arm to initiate exploration.^[19] The session lasts 5 minutes and is conducted under dim red lighting (around 20–40 lux) to simulate low-stress conditions and prevent visual overstimulation.^[21] During this time, the animal is allowed to explore freely without interference, and the trial is terminated early only if the animal falls from the maze, which is a rare occurrence with proper barriers.^[22] Data collection occurs via overhead video recording or manual observation from an adjacent room to avoid influencing behavior, capturing the animal's movements across the arms for later analysis.^[4]^[15] Following the trial, the animal is promptly returned to its home cage, and the apparatus is cleaned with 70% ethanol or a similar odor-neutralizing solution to eliminate olfactory cues between subjects.^[16]^[22] In designs involving repeated measures, a minimum 24-hour washout period is observed to allow recovery and prevent carryover effects.^[17]

Behavioral Assessment

Key Measures

The primary measures of anxiety in the elevated plus maze focus on the animal's exploration of the open arms, which are thought to evoke an unconditioned fear response due to their exposure. The percentage of time spent in the open arms is calculated as (% open arm time = (time in open arms / total time on the maze) × 100), where total time is typically 5 minutes.^[23] Similarly, the percentage of entries into the open arms is determined by (% open arm entries = (open arm entries / total arm entries) × 100), with an arm entry defined as all four paws crossing into the arm.^[2] These metrics are the most commonly used indicators, as reduced open arm activity correlates with heightened anxiety-like behavior.^[23] Secondary measures provide context for overall activity and complementary risk assessment. Total arm entries serve as an indicator of locomotor activity, calculated simply as the sum of entries into both open and closed arms, helping to distinguish anxiety effects from general movement changes.^[23] Time spent in closed arms is the complement to open arm time, often reported as (time in closed arms / total time) × 100, reflecting preference for protected spaces.^[2] Head-dipping frequency in the open arms, defined as the number of times the animal dips its head below the level of the maze floor while in an open arm, assesses exploratory risk-taking behavior.^[23] Ethological endpoints capture more nuanced anxiety-related behaviors beyond arm preference. Protected head-dips occur from the closed arms or central platform, indicating cautious exploration, while unprotected head-dips happen from open arms and suggest greater risk tolerance.^[23] Stretched attend postures (SAPs) are recorded as the frequency of forward elongations of the body toward an area of interest without moving the hind paws, often classified as protected (from closed areas) or unprotected (from open areas); higher frequencies signal anxiety-driven risk assessment.^[23] Grooming bouts, measured as the number or duration of self-grooming episodes, increase under anxiogenic conditions and serve as an additional indicator of emotional distress.^[23] Scoring in the elevated plus maze can be automated or manual to ensure reliability. Automated systems, such as ANY-maze software, use video tracking to detect center line crossings for arm entries and time spent, providing precise zone-based metrics with minimal observer bias.^[24] Manual coding, often via video playback, is employed for subtle ethological behaviors like SAPs and grooming, where observers tally occurrences in real-time or post hoc using standardized definitions.^[23] Under baseline conditions, rodents typically exhibit low open arm exploration, with anxious phenotypes spending less than 20% of time in open arms.^[23] Anxiolytic drugs, such as benzodiazepines, reliably increase open arm time and entries by 20-50%, demonstrating the assay's sensitivity to anti-anxiety effects.^[2]

Interpretation of Results

In the elevated plus maze, low time spent and fewer entries into the open arms are interpreted as indicators of high anxiety-like behavior, reflecting the rodent's aversion to the exposed, elevated areas due to innate fear of open spaces and heights.^[1] This avoidance is positively correlated with activation of the hypothalamic-pituitary-adrenal (HPA) axis, as evidenced by elevated corticosterone levels in animals exhibiting reduced open arm exploration, linking the behavioral response to physiological stress mechanisms.^[25] However, interpretations must account for confounding factors such as locomotor activity, where high overall exploration can artificially inflate open arm entries without necessarily indicating reduced anxiety; researchers often normalize open arm measures by total arm entries or distance traveled to isolate anxiety-specific effects from general motility changes.^[5] ^[26] Pharmacological validation supports the maze's sensitivity to anxiolytics, with benzodiazepines like diazepam (administered at 1-2 mg/kg) reliably increasing time and entries in open arms by enhancing GABAergic inhibition and reducing fear responses.^[27] ^[28] Similarly, 5-HT1A receptor agonists such as buspirone (3-10 mg/kg) promote open arm exploration, confirming the test's predictive validity for serotonin-modulating anxiolytics.^[29] ^[30] Strain and sex differences further influence interpretations, with BALB/c mice displaying higher anxiety-like behavior—characterized by lower open arm time—compared to C57BL/6 mice, which exhibit greater baseline exploration and resilience to stressors.^[31] ^[32] Females often show higher open arm activity than males, particularly during proestrus, suggesting lower anxiety or enhanced exploratory drive influenced by ovarian hormones.^[33] ^[1] Statistical analyses typically employ analysis of variance (ANOVA) for group comparisons of open arm metrics, revealing moderate to large effect sizes (Cohen's d ≈ 0.5-1.0) for anxiolytics, which quantify the magnitude of behavioral shifts while controlling for variability in baseline responses.^[34] ^[35] Despite these insights, interpretations are limited by the test's non-specificity as a pure anxiety measure, as open arm avoidance can also reflect responses to novelty, risk assessment, or decision-making processes rather than anxiety alone, necessitating complementary assays for comprehensive phenotyping.^[5] ^[36]

Applications

In Animal Research

The elevated plus maze (EPM) serves as a cornerstone in preclinical animal research for evaluating anxiety-like behaviors, particularly in rodents, where it facilitates the screening of potential anxiolytic compounds. It is routinely employed to test the efficacy of pharmacological agents such as selective serotonin reuptake inhibitors (SSRIs) and gamma-aminobutyric acid (GABA) receptor agonists, including benzodiazepines like diazepam, which typically increase the time spent in open arms, indicating reduced anxiety.^[1]^[37] This paradigm has been validated for modeling anxiety disorders such as generalized anxiety disorder (GAD) and post-traumatic stress disorder (PTSD) in rodent models, where anxiolytics reverse stress-induced reductions in exploratory behavior.^[38] For instance, in PTSD rodent models, the EPM detects impairments in open arm exploration following chronic stress exposure, supporting its role in high-throughput drug discovery.^[39] In genetic models of anxiety, the EPM is instrumental for phenotyping mutant rodents, revealing heightened anxiety-like behaviors in serotonin transporter (5-HTT) knockout mice, which spend significantly less time in open arms compared to wild-type controls.^[40] Similarly, 5-HT1A receptor knockout mice exhibit reduced exploratory activity and increased anxiety in the EPM, underscoring the role of serotonergic pathways in emotional regulation.^[41] These findings highlight the EPM's sensitivity to genetic manipulations, enabling researchers to dissect heritability of anxiety traits and evaluate gene-environment interactions. Neurobiological investigations using the EPM often target key brain regions like the amygdala and prefrontal cortex (PFC), where lesions or optogenetic manipulations alter anxiety outcomes. For example, optogenetic stimulation of the medial PFC in stressed rodents increases open arm time, demonstrating its anxiolytic potential via projections to the basolateral amygdala.^[42]^[43] The basolateral amygdala, in particular, modulates avoidance behaviors in the EPM, with inhibitory optogenetics reducing anxiety-like responses, thus linking circuit-level activity to behavioral phenotypes.^[44]^[45] Developmental studies leverage the EPM to examine how early life stress influences anxiety trajectories across ages in rodents. Prenatal stress exposure leads to age-dependent increases in anxiety-like behavior, with affected offspring showing decreased open arm entries in juvenile and adult stages.^[46] In aging models, while the EPM remains applicable without strict age restrictions, chronic early stress accelerates anxiety phenotypes in older rodents, reflecting cumulative neurodevelopmental impacts.^[47]^[48] Beyond rodents, the EPM has been adapted for comparative translational research in non-rodent species to enhance cross-species validity. In zebrafish, plus-maze variants assess anxiety-like avoidance, with anxiolytics increasing exploration of open areas, supporting its utility in high-throughput screening for conserved mechanisms.^[49]^[50] For primates like monkeys, analogous elevated mazes evaluate anxiety in social and stress contexts, bridging rodent findings to higher-order models.^[51] Key empirical findings from EPM studies reinforce its reliability: chronic stress paradigms, such as repeated restraint, significantly reduce open arm time in rodents, mimicking pathological anxiety states.^[52] Conversely, exercise interventions, including treadmill running, increase open arm duration and entries, promoting anxiolytic effects that validate behavioral therapies.^[53]^[54] These outcomes, measured as percentages of total session time (typically 5 minutes), provide quantifiable benchmarks for anxiety modulation.^[1]

In Human Studies

The elevated plus maze has been adapted for human use primarily through virtual reality (VR) implementations developed in the 2010s, allowing participants to navigate a 3D representation of the maze via head-mounted displays to assess approach-avoidance behaviors related to anxiety.^[55] These adaptations translate the rodent paradigm into an ecologically valid human assay by simulating the maze's height and open/closed arms in immersive environments, often combining virtual elements with real-world cues for enhanced presence.^[55] In typical protocols, participants undergo a 5- to 10-minute immersion in the VR maze, freely exploring after a brief baseline period, while metrics such as time spent in virtual open arms, entries into those arms, latency to enter, gaze direction via eye-tracking, and physiological responses like heart rate variability are recorded.^[56] Gaze aversion from open areas and increased heart rate in those zones serve as indicators of anxiety-like avoidance.^[55] Validation studies demonstrate that VR elevated plus maze performance correlates moderately with self-report anxiety measures, such as the State-Trait Anxiety Inventory (STAI), particularly for trait anxiety (r ≈ 0.30) and acrophobia questionnaires (r ≈ -0.40 to -0.51 for open arm avoidance).^[56] The paradigm is sensitive to pharmacological interventions, with anxiolytics like lorazepam reducing open arm avoidance (t = 1.86, P = 0.036) and anxiogenics like yohimbine increasing it (t = 1.86, P = 0.037), mirroring rodent responses.^[55] Applications include investigating phobias (e.g., acrophobia), risk-taking behaviors, and decision-making under uncertainty, with use in both healthy volunteers and individuals with anxiety disorders or comorbid conditions like substance use problems.^[56]^[57] For instance, it has been employed in clinical trials to evaluate anxiolytic efficacy and in observational studies of young adults with problematic alcohol or cannabis consumption to detect heightened anxiety and craving responses.^[55]^[57] Compared to animal models, the human VR version offers ethical advantages by avoiding physical risk and enabling concurrent verbal reports of subjective anxiety, as shown in mixed reality setups that enhance ecological validity through real-virtual integration.^[55] However, limitations include simulator sickness affecting some participants (Simulator Sickness Questionnaire scores ≈ 20-21), potential habituation over repeated exposures, and reduced ethological fidelity relative to the rodent paradigm due to the virtual nature of threats.^[55]

Variations and Modifications

Elevated Zero Maze

The elevated zero maze is a circular variant of the elevated plus maze designed specifically for assessing anxiety-like behaviors in rodents by promoting more natural exploratory patterns. Introduced by Shepherd et al. in 1994, this apparatus addresses limitations in the traditional plus maze, such as interpretive ambiguity from the central platform and potential biases in corner exploration, thereby facilitating uninterrupted movement and enhanced ethological analysis.^[58] The design features a circular platform with a diameter of approximately 50 cm for mice and 105 cm for rats, elevated approximately 50 cm above the ground, divided into four equal quadrants alternating between two open (unprotected) and two enclosed (walled) sections positioned 90 degrees apart. Unlike the plus maze, it lacks a central platform, eliminating decision points that could confound behavior. The open quadrants are typically devoid of walls or ledges, while closed quadrants have opaque walls (15-40 cm high, depending on species) to provide shelter, constructed from materials like black Perspex or wood to minimize reflections and neophobia.^[58]^[59] The experimental procedure mirrors the plus maze in simplicity, involving a single 5-minute trial where the animal is placed in a closed quadrant facing the inner wall to initiate exploration. Sessions are conducted under dim lighting (e.g., 40-60 lux) in a quiet room to reduce external stressors, with the apparatus wiped clean between trials to eliminate olfactory cues. Automated video tracking software is often employed for precise data collection, enhancing reliability.^[58]^[60] Key behavioral measures include the percentage of time spent in open quadrants, which inversely correlates with anxiety levels—higher times indicate reduced anxiety—and the number of transitions between quadrants, serving as a proxy for locomotor activity and exploratory drive. These metrics share conceptual overlap with the plus maze's open arm time but benefit from the zero maze's continuous design for clearer interpretation. Additional ethological endpoints, such as head dips over open edges or stretched attend postures from closed areas, provide nuanced insights into risk assessment.^[58] Advantages of the elevated zero maze include the removal of central hesitation points, which promotes smoother transitions and reduces neophobia compared to the plus maze, leading to more consistent and quantifiable data across sessions. Its circular layout also facilitates automated tracking by simplifying path analysis and minimizing edge effects, making it particularly suitable for high-throughput studies. Furthermore, it exhibits less initial aversion to open areas, improving sensitivity to subtle anxiolytic effects.^[58]^[61] This apparatus is primarily used to evaluate anxiety in rats and mice, with pharmacological validation demonstrating sensitivity to the same anxiolytics (e.g., benzodiazepines like diazepam) and anxiogenics (e.g., mCPP) as the plus maze, confirming its reliability for preclinical drug screening and neurobehavioral research.^[58]

Elevated T-Maze

The elevated T-maze (ETM) is a T-shaped apparatus consisting of three arms of equal length, typically 50 cm long and 10 cm wide, elevated 50 cm above the floor (dimensions adjusted for species). One arm serves as the enclosed stem with lateral walls 40 cm high, while the two opposing arms are open, featuring only a low ledge (about 0.5 cm high) along their edges to prevent falls. This configuration exploits the animal's innate aversion to elevated open spaces, similar to principles underlying the original elevated plus maze.^[62] The ETM was developed in the early 1990s by researchers including M.B. Viana, C. Tomaz, and F.G. Graeff as an adaptation of traditional T-mazes to study both anxiety and memory processes in rodents. It builds on earlier maze designs but incorporates elevation to enhance ethological validity for anxiety assessment, allowing differentiation between conditioned and unconditioned fear responses. The task has since been validated in rats and mice, with refinements focusing on its sensitivity to anxiolytic and serotonergic compounds.^[62]^[63] The procedure involves two main phases conducted in a dimly lit room to minimize external stimuli. In the training phase for inhibitory avoidance, the rodent is placed at the distal end of the enclosed stem arm, and the latency to exit this arm by entering one of the open arms is recorded over three consecutive trials, with inter-trial intervals of about 30 seconds. This phase induces learning through repeated exposure to the aversive open arms. Approximately 24 to 96 hours later, in the test phase, the animal is again placed in the enclosed arm, and the latency to re-enter an open arm is measured as a single trial, reflecting retention of the avoidance behavior. A separate one-way escape task follows, where the animal is placed at the end of an open arm, and the latency to escape back to the enclosed arm is recorded in one trial.^[62]^[63] Key measures include inhibitory avoidance latency, defined as the time taken to leave the enclosed arm during training and testing, which increases across trials due to learned aversion to the open arms and indicates conditioned fear. Escape latency, the time to retreat from an open arm to the enclosed one, remains consistently short and stable, serving as an index of unconditioned fear or panic-like responses. These latencies are typically capped at 300 seconds to account for non-responders.^[63] The ETM primarily models conditioned fear associated with generalized anxiety through the inhibitory avoidance component, while the escape task captures unconditioned fear akin to panic responses. It is particularly sensitive to drugs modulating the serotonin (5-HT) system, such as selective serotonin reuptake inhibitors and 5-HT1A agonists, which reduce avoidance latencies without affecting escape, suggesting a role for 5-HT in inhibitory control of fear. This dual assessment enables evaluation of serotonergic interventions in anxiety disorders.^[63] Unlike the elevated plus maze, which relies on free exploration and choice in a single session, the ETM employs a forced-choice paradigm with positioned placements and a multi-trial training component to incorporate learning and memory elements, allowing separation of innate anxiety from acquired avoidance.^[63]

Other Specialized Variants

The plus-maze discriminative avoidance test (PMDAT) modifies the standard elevated plus maze by incorporating an aversive stimulus in one of the closed arms during a training phase, allowing simultaneous assessment of conditioned avoidance learning and anxiety-like behavior. In the original paradigm, mice or rats are exposed to light and noise as the aversive cue in one enclosed arm, leading to avoidance of that arm in subsequent tests while still exhibiting open-arm avoidance indicative of anxiety. Later adaptations, such as those using mild footshock (0.1–0.3 mA for 1 s) in the target closed arm, enhance the model's sensitivity to fear memory reactivation and interactions with stress hormones like corticosterone, though metyrapone can block hormonal rises without affecting memory reinstatement. This variant demonstrates anxiolytic sensitivity to drugs like chlordiazepoxide, which increases open-arm time but impairs avoidance memory, while maintaining utility for studying state-dependent learning effects of substances like caffeine or morphine. The multivariate concentric square field (MCSF) test extends the plus-maze concept into a more complex arena composed of nested concentric squares featuring distinct zones—such as sheltered (dark, enclosed), open (dimly lit), elevated (illuminated), and transitional corridors with a hole board—to profile multiple behavioral dimensions including exploration, risk-taking, risk assessment, and shelter-seeking in rodents. Developed for ethoexperimental analysis, the MCSF uses principal component analysis to differentiate strain, sex, and stress influences on behavior, with food-deprived animals showing increased risk-taking (e.g., open-zone entries) and lactating females prioritizing pup retrieval in safe zones. Unlike the binary open-closed arms of the plus maze, the MCSF's multi-zone design reveals nuanced anxiety profiles, as anxiolytics like diazepam increase entries into risky areas while introducing confounds related to general activity levels. In human studies, virtual reality (VR) adaptations of the elevated plus maze incorporate mixed-reality elements to simulate the rodent paradigm, blending physical structures (e.g., a wooden platform) with immersive virtual environments like rocky landscapes to evoke height-induced anxiety without real elevation risks. A 2017 mixed-reality version demonstrated robust open-arm avoidance in participants, with increased subjective anxiety ratings, cortisol, and alpha-amylase on open arms compared to closed ones (t_{99} = -11.89, P < 0.001), validating cross-species translational potential. Pharmacological validation showed lorazepam reducing avoidance and yohimbine enhancing it, though interactive virtual cues (e.g., environmental feedback) can introduce variability in arousal responses not present in static rodent setups.^[55] Automated variants of the elevated plus maze integrate RFID tracking systems, where rodents are implanted with subcutaneous tags to enable precise, non-invasive identification and monitoring of individual movements across arms without relying on video-based color marking that may alter anxiety. These systems facilitate long-term or group testing by logging zone entries, latency, and velocity in real-time, improving data reliability for anxiolytic screening while minimizing handler-induced confounds. Light-dark hybrids, such as combined enclosures in one arm, further refine specificity for photophobia-related anxiety but risk overcomplicating interpretation due to competing light-aversion drives. Overall, these specialized variants enhance fear learning specificity (e.g., PMDAT) or multi-trait assessment (e.g., MCSF) and retain sensitivity to anxiolytics, though each introduces unique confounds like memory interference or elevated activity bias.

Criticisms and Limitations

Validity and Reliability Issues

The elevated plus maze (EPM) demonstrates pharmacological validity through its sensitivity to classical anxiolytics, such as benzodiazepines, which reliably increase the time spent in open arms by reducing anxiety-like avoidance behaviors in rodents.^[23] However, this sensitivity is inconsistent for antidepressants, with acute administration often failing to produce anxiolytic effects and sometimes even eliciting anxiogenic responses, limiting its utility for screening non-GABAergic compounds.^[64] Furthermore, the test has shown failures in detecting anxiolytic potential for certain novel compounds, particularly those targeting non-traditional pathways, due to procedural confounds like handling stress.^[65] Ethological validity is supported by the EPM's emulation of natural predator avoidance, as rodents instinctively shun open, elevated spaces akin to exposed terrains in the wild, with behaviors like stretched attend postures reflecting risk assessment.^[23] Nonetheless, this is confounded by the novelty of the apparatus, which elicits exploration-driven responses that may mask true anxiety, and the test performs poorly in modeling chronic anxiety states, better suiting acute, state-like fear rather than sustained disorders.^[65] Predictive validity for human anxiety translation is moderate, particularly for GABAergic agents, but it overestimates efficacy for non-GABA drugs that later fail in clinical trials.^[66] Reliability issues arise from significant inter-laboratory variability due to inconsistent protocols, and strain-dependent outcomes, such as Swiss-Webster mice exhibiting higher open arm exploration than Wistar rats under similar conditions.^[65]^[67] A 1995 review by Dawson and Tricklebank highlighted contradictory results across pharmacological studies, attributing them to unstandardized testing.^[66] To mitigate variance, standardized lighting (e.g., 100-200 lux) and gentle handling procedures have been recommended.^[65]

Methodological Challenges

Rodents exhibit rapid habituation to the elevated plus maze, leading to a one-trial tolerance effect where repeated testing within days invalidates anxiety-like behavior measures due to decreased exploratory activity in open arms.^[12] This phenomenon arises because initial exposure induces lasting changes in behavior, such as reduced entries into aversive open arms on subsequent trials, confounding longitudinal assessments.^[12] Several confounds compromise the interpretation of results in the elevated plus maze, including locomotor activity that can mask true anxiety effects; for instance, stimulants like amphetamine may increase open-arm entries not through anxiolysis but via heightened general activity.^[1] Additionally, neophobia during the first minute of testing often skews data, as animals initially avoid open arms due to novelty rather than anxiety, necessitating exclusion of this period from analyses.^[1] Environmental factors introduce significant variability in elevated plus maze outcomes, with lighting levels (typically 20-50 lux in the center) directly influencing arm preference; brighter illumination increases anxiety-like avoidance, while inconsistent lux can alter baselines across experiments.^[68] Noise and residual odors from prior trials similarly disrupt behavior, prompting recommendations for quiet, odor-neutral rooms and thorough cleaning between sessions to ensure controlled conditions.^[69] Manual scoring exacerbates subjectivity, as observer bias in identifying arm entries or protected head-dips leads to inter-rater inconsistencies; automated video tracking systems mitigate this by providing objective, reproducible metrics.^[70] Recent studies highlight methodological inconsistencies influenced by apparatus characteristics in young adult and old rodents in the elevated plus maze, where older mice may show higher open-arm exploration in certain setups.^[71] Sex differences remain underreported, with many studies failing to stratify analyses by sex, reducing generalizability.^[72] As of 2025, research has proposed advanced tracking methods, such as measuring exploration depth and timing, to resolve inconsistencies in anxiety-like behavior assessment.^[73] To address these challenges, incorporating risk assessment behaviors like stretch-attend postures (SAPs) offers a more nuanced alternative to traditional arm-entry metrics, as these ethological measures better isolate anxiety from locomotion.^[1] Employing multi-test batteries, such as combining the elevated plus maze with open-field assays, enhances robustness by cross-validating results and minimizing single-test confounds.^[74]

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In practice, the test consists in placing the subjects individually in the centre of the apparatus facing the open arm and allow to explore the maze freely for ...Missing: placement | Show results with:placement
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Elevated plus maze - Bio-protocol
Elevated plus maze (EPM) testing was performed ... Testing was conducted under 20 lux red light. The ... Preprint Protocol Preparation Guidelines. For ...
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None
### Experimental Procedure Details for Elevated Plus Maze
[20]
https://www.sciencedirect.com/topics/%5Bneuroscience
[21]
Elevated Plus-maze - ANY-maze
The key to automating the elevated plus-maze is to detect zone entries and exits with precision · Precision tracking. ANY-maze's whole body tracking allows you ...
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Influence of reactivity to novelty and anxiety on hypothalamic ...
min in a circular corridor, and as high or low anxiety (HA versus LA) as a function of the time spent in the open arms of the elevated plus-maze. Then, the ...
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Utility of ethological analysis to overcome locomotor confounds in ...
Recently, it has been shown that ethological measures of "risk assessment", such as stretched-attend postures and head-dipping, are sensitive indicators of drug ...Missing: sources | Show results with:sources
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Validation of open:closed arm entries in an elevated plus-maze as a ...
A novel test for the selective identification of anxiolytic and anxiogenic drug effects in the rat is described, using an elevated + -maze consisting of two ...
[25]
Diazepam Reduces Escape and Increases Closed-Arms Exploration ...
Apr 1, 2019 · The elevated plus-maze (EPM) is a widely used test to evaluate anxiety-related behaviors and to detect anxiolytic properties of compounds in ...Abstract · Introduction · Results · Discussion
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An evaluation of the elevated plus-maze test using the novel ...
These results suggest that an agonist action at postsynaptic 5-HT1A receptors mediates the anxiogenic-like effects of buspirone in the elevated plus-maze test ...
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Role of 5-HT1A Receptor in the Anxiolytic-Relaxant Effects of ... - MDPI
Elevated Plus Maze (EPM) and Open field (OF) tests were used to measure relaxant-anxiety effects, whereas Rotarod test was used to investigated motor impairment ...Role Of 5-Ht1a Receptor In... · 2. Results · 2.2. Elevated Plus Maze Test<|separator|>
[28]
Evaluation of the elevated plus-maze and open-field tests ... - PubMed
The factorial scores of BALB/c and C57BL/6 mice were analysed by MANOVAs, which showed significant effects of both the strain and test used. Our results ...
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Strain and Sex Differences in Anxiety-Like and Social Behaviors in ...
C57BL/6J mice showed lower levels of anxiety-like behavior than female and male BALB/c mice in the open field and elevated plus maze tests. Sexual differences.<|separator|>
[30]
Sex differences in the elevated plus-maze test and large open field ...
Mar 5, 2021 · The females spent more time on the open arms of the EPM and made more open arms entries than the males. The females also spent more time in the ...
[31]
Reliability of common mouse behavioural tests of anxiety
Meta-analyses of effect sizes of treatments showed that only two out of 17 commonly used test measures reliably detected effects of anxiolytic compounds. We ...
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[PDF] Effects of Diethylene Glycol on Cognitive Abilities in Rats - Brieflands
Jul 31, 2025 · effect size of Cohen's d ≈ 1.17. ... Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of ...
[33]
Preclinical animal anxiety research – flaws and prejudices - PMC
The current tests of anxiety in mice and rats used in preclinical research include the elevated plus‐maze (EPM) or zero‐maze (EZM), the light/dark box (LDB) ...
[34]
The Gerbil Elevated Plus-Maze I: Behavioral Characterization and ...
Feb 22, 2002 · All studies testing for anxiolytic-like effects were conducted under high light conditions (500-Lux). The drugs tested for anxiolytic-like ...
[35]
Rodent models of post-traumatic stress disorder: behavioral ... - Nature
May 6, 2020 · Walf, A. A. & Frye, C. A. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat. Protoc. https://doi.org ...Missing: aging | Show results with:aging
[36]
Animal models for screening anxiolytic-like drugs: a perspective
Apr 1, 2022 · The elevated plus maze test, whatever the modalities of use, is still an important tool for discovery of anxiolytics; agomelatine, for example, ...<|separator|>
[37]
Opposing alterations in anxiety and species-typical behaviours in ...
5-HTT overexpressing mice displayed reduced anxiety-like behaviour whilst 5-HTT knockout mice showed increased anxiety-like behaviour, compared to wildtype ...
[38]
Serotonin receptor 1A knockout: An animal model of anxiety-related ...
In summary, our results indicate that 5-HT1A knockout mice display less exploratory activity, more anxious-like behaviors, and possibly less behavioral despair ...
[39]
Antidepressant Effect of Optogenetic Stimulation of the Medial ...
Dec 1, 2010 · Optogenetic stimulation of mPFC exerted potent antidepressant-like effects, without affecting general locomotor activity, anxiety-like behaviors, or social ...
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Identification of a prefrontal cortex-to-amygdala pathway for chronic ...
May 6, 2020 · The mouse anxiety-like behaviors were tested with elevated plus maze (EPM) and open field test (OFT) (Fig. 5a–c) and the Pr was measured with ...
[41]
Bidirectional modulation of anxiety-related and social behaviors by ...
May 3, 2016 · The basolateral amygdala (BLA) and the medial prefrontal cortex (mPFC) modulate anxiety and social behaviors. It remains to be elucidated, ...
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Amygdala Function and Experimental Manipulation - Maze Engineers
May 2, 2018 · The amygdala coordinates autonomic, endocrine and behavioral responses to stimuli from the environment, and most particularly it integrates inputs that stem ...Amygdala Subregions · Amygdala In Maze Learning · Optogenetics For Testing The...
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Behavior ontogeny in the elevated plus-maze: prenatal stress effects
Discussion. The present study showed that prenatal stress led to age-dependent effects on anxiety as evaluated in the elevated plus-maze. Prenatal stress ...Missing: aging rodents
[44]
A Review of Behavioral Tests to Evaluate Different Types of Anxiety ...
Abstract: Behavioral tests are very useful to understand the Neuro-psychotic disease and also helpful in finding the treatment of the particular disease.
[45]
Behavior in the elevated plus maze is differentially affected by ...
We assessed anxiety behavior on an elevated plus maze (EPM) in juvenile rats around 3 weeks of age under diverse testing conditions.
[46]
Associative learning performance is impaired in zebrafish (Danio ...
Here we show that zebrafish exhibit good learning performance in a task adapted from the mammalian literature, a plus maze in which zebrafish are required to ...
[47]
Measuring anxiety in zebrafish: A critical review - ScienceDirect.com
We review some procedures which have been used to study anxiety, fear or related processes in zebrafish, using the validity framework.
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The future of zebrafish in behavioral neuroscience - ScienceDirect
Zebrafish is becoming increasingly popular in behavioral neuroscience. This review explores theoretical and practical reasons for it.Missing: elevated | Show results with:elevated
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Time spent on the open arms of the elevated plus maze as a ...
Time spent on the open arms of the elevated plus maze as a measure of anxious-like behavior in male and female C57BL/6 naive, chronic stress, nonstressed post- ...
[50]
Influence of Chronic Moderate Sleep Restriction and Exercise ... - NIH
Exercise training elicited a significant increase in time spent in the open arms of the maze, a result that is indicative of reduced anxiety. These data are ...Missing: key | Show results with:key
[51]
Treadmill exercise alleviates stress-induced anxiety-like behaviors ...
Oct 31, 2018 · Anxiety-like behaviors were determined by open field test and elevated plus-maze test. The expressions of c-Fos and neuronal nitric oxide ...<|control11|><|separator|>
[52]
https://www.researchgate.net/figure/Time-spent-on-the-open-arms-of-the-elevated-plus-maze-as-a-measure-of-anxious-like_fig2_325375856
[53]
Effects of Acrophobic Fear and Trait Anxiety on Human Behavior in a ...
Apr 19, 2021 · The Elevated Plus-Maze (EPM) is a well-established apparatus to measure anxiety in rodents, i.e., animals exhibiting an increased relative time ...Missing: primary | Show results with:primary
[54]
https://www.e-jer.org/journal/view.php?number=2013600570
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https://doi.org/10.1186/s12915-017-0463-6
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Zero Maze - Maze Engineers - ConductScience
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 ...
[57]
https://doi.org/10.1016/j.ynstr.2023.100564
[58]
Anxiety in the elevated zero maze - Noldus
Apr 6, 2022 · The Elevated Zero Maze (EZM) is a ring-shaped arena with open and closed quadrants, used to test anxiety-like behavior by removing the center ...
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https://maze.conductscience.com/portfolio/zero-maze/
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Effects of acute fluoxetine, paroxetine and desipramine on rats ...
Jan 25, 2007 · The elevated plus-maze exhibits good sensitivity for detecting anxiogenic effects of antidepressant drugs and the conventional parameters ...
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https://noldus.com/blog/measuring-anxiety-elevated-zero-maze
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https://doi.org/10.1016/0091-3057(94)90067-1
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Effects of test experience, closed-arm wall color, and illumination ...
Feb 15, 2021 · Increased open arm time and entries are conventionally interpreted as decreased anxiety ... Rodents normally avoid open arms in an elevated plus ...
[64]
Standardization of elevated plus maze - Noldus
Jul 8, 2021 · Standardization of, or standardizing, anxiety in rodents in the elevated plus maze ... A reversed cycle however requires working under red-light ...Missing: protocol | Show results with:protocol
[65]
Full article: Computer-Assisted Scoring of the Elevated Plus Maze
We report here the development of a program to facilitate EPM scoring. EPMscore.exe is freely available as a standalone DOS executable program.
[66]
Effect of apparatus characteristics on anxiety-like behavior in young ...
Initial experiments using the EPM assay to assess anxiety-like behaviors were performed by Handley and Mithani (1984) in rats using an apparatus with 45 cm long ...
[67]
Sex-specific behavioral changes in middle-aged mice - Nature
Jul 1, 2025 · Our findings revealed that both sexes presented increased anxiety-like behavior and impaired spatial recognition memory during middle age compared to young ...Missing: inconsistencies | Show results with:inconsistencies
[68]
Test, rinse, repeat: A review of carryover effects in rodent behavioral ...
The elevated plus maze (EPM) and elevated zero mazes (EZM) are widely used to evaluate anxiety-like behavior in rodents. The EPM consists of a plus-shaped maze ...Missing: invalidation | Show results with:invalidation