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Centrifugation

Ultracentrifugation Basics and Applications

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Introduction

Ultracentrifugation is a specialized technique used to spin samples at exceptionally high speeds. Current ultracentrifuges can spin to as much as 150 000 rotations per minute (rpm) (equivalent to 1 000 000 g) (Biocompare, 2019b). However, extreme centrifugal forces may cause overheating, so to avoid sample damage, ultracentrifuges are equipped with vacuum systems that keep a constant temperature in the centrifuge’s rotor (Biocompare, 2019b).

Centrifugation, and ultracentrifugation, is nowadays, at the core of the laboratory routine. Benchtop centrifuges are essential devices in any biology or chemistry laboratory, and they are used on a day-to-day basis in a wide range of experimental protocols, from concentrating solutions to isolating cells and subcellular components. Ultracentrifugation widened the applications of benchtop centrifugation, allowing the isolation of smaller sized particles, and the study of purified molecules and molecular complexes (Ohlendieck & Harding, 2017). In biology, the development of ultracentrifugation in the early 1900s, widened the possibilities of scientific research to the subcellular level, allowing for the differential separation of cellular components, such as organelles, lipid membranes, and even to purify proteins and ribonucleic acids (DNA and RNA).

The Principle of Ultracentrifugation

The basis of ultracentrifugation is the same as normal centrifugation: to separate the components of a solution based on their size and density, and the density (viscosity) of the medium (solvent) (Ohlendieck & Harding, 2017).  As a general principle, (ultra)centrifugation abides by the following rules (Ohlendieck & Harding, 2017):

  • the denser a biological structure is, the faster it sediments in a centrifugal field
  • the more massive a biological particle is, the faster it moves in a centrifugal field
  • the denser the biological buffer system is, the slower the particle moves in a centrifugal field
  • the greater the frictional coefficient (i.e., the friction between the component and the neighboring environment) is, the slower a particle moves
  • the greater the centrifugal force is, the faster the particle sediments
  • the sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal.
Centrifugation Versus Ultracentrifugation

The use of ultracentrifugation over centrifugation (and vice-versa) denotes basic differences between the two techniques. Fundamental differences between centrifugation and ultracentrifugation include:

  1. Spinning velocity, and therefore, the centrifugal force applied to the samples. The rotor of an ultracentrifuge can spin as high as 1 000 000 x g, while most common benchtop centrifuges are limited to 65 000 x g (Biocompare, 2019a, 2019b). This brings up the second basic difference:
  2. Refrigeration and vacuum systems, are mandatory in ultracentrifuges. Because of the extremely high spinning speed, ultracentrifuges are always equipped with vacuum and refrigeration systems, to avoid sample and/or device damage due to frictional force and overheating. In benchtop centrifuges, these two systems are optional, with the most simple centrifuges, like mini microcentrifuges, displaying none.
  3. Type of pellet that is produced from sample fractionation: because ultracentrifuges can achieve much higher spinning speeds, the type of sediment (pellet) that results from one or the other is also different, with ultracentrifugation allowing for the isolation of smaller particles than benchtop centrifugation. In biology labs, subcellular fractionation to separate cytosolic contents (such as whole cytosol, mitochondria, or chloroplasts) from cell nuclei can be attained by benchtop centrifuges. However, to isolate smaller components such as ribosomes and small vesicles, higher centrifugal forces, only possible with ultracentrifuges, are necessary (Momen-Heravi, 2017; Ohlendieck & Harding, 2017).
Types of Ultracentrifugation: Analytical Versus Preparative

There is, currently on the market, a wide variety of ultracentrifuges. The choice among different brands and models must consider the type of experimental applications to be performed, the availability of different rotors (making it possible to adapt the ultracentrifuge to different experimental settings), and the temperature range. Some ultracentrifuges further offer remote monitoring and control, and password-protection (Biocompare, 2019b).

It is important to ask: For which experimental setting do I need an ultracentrifuge? In that sense, two types of ultracentrifuges are available: analytical and preparative. Analytical ultracentrifugation is used in the study of purified macromolecules or supramolecular assemblies, while preparative ultracentrifugation is used in the actual separation of tissues, cells, subcellular components, and other biochemically interesting particles (Ohlendieck & Harding, 2017).

Analytical ultracentrifuges are equipped with optical detection systems that allow the researcher to follow the centrifugation process in real-time. These systems may use ultraviolet (UV) light absorption or refracting index interference (RII) optical detection systems (ultracentrifuges may be equipped with one or both types of optical systems) (Cole, 2009). While UV detection directly measures the absorbance (abs) of a substance at a specific wavelength, RII measures changes in the refraction index (radiation direction) of a given substance, compared to the solvent it is dissolved in (Ohlendieck & Harding, 2017). The purpose of analytical centrifugation is different from other types of centrifugation. Although component isolation is possible with analytical centrifugation, the goal of this technique is to obtain data to characterize the sample that is spun (sedimentation velocity, viscosity, concentration, etc.). With analytical centrifugation, it is possible to follow the variations in sample concentration as a function of the applied centrifugal force. This technique is used in two main experimental settings: sedimentation velocity and sedimentation equilibrium studies, which are key in macromolecular characterization (Ohlendieck & Harding, 2017). Results from sedimentation velocity experiments provide data that are used to calculate the molecular size (molecular weight), shape, and molar masses of new chemical molecules, ribonucleic acids, proteins, and others (Cole, 2009; Ohlendieck & Harding, 2017).

Preparative ultracentrifuges are mostly used to process biological samples for further analysis. The most common application of preparative ultracentrifugation is in tissue and subcellular fractionation, to isolate increasingly smaller components of the biological samples (Jasinski, Schwartz, Haque, & Guo, 2015; Martin et al., 2018; Momen-Heravi, 2017; Ohlendieck & Harding, 2017; Wasan, Cassidy, Kennedy, & Peteherych, 2001). For that, two main centrifugation methods are used: differential and density-gradient centrifugation.

  1. Differential centrifugation is used to separate the components of a solution based on differences in the sedimentation rate of the different components of the mixture. As explained above (see section 2: The Principle of Ultracentrifugation), the sedimentation properties of a substance depend on its size and density but also on the density of the solvent. In medical and biology labs, crude tissue homogenates containing organelles, membrane vesicles, and other structural fragments are divided into different fractions by the stepwise increase of the applied centrifugal field. Furthermore, differential centrifugation is also routinely used in the isolation of non-living substances, like nanoparticles, colloids, and viruses (Long, Borsa, & Sargent, 1976). The general principle of differential centrifugation is outlined in Figure 1.

Figure 1: General principle of differential centrifugation, applied to subcellular fractionation (image credit: (Kumar))

Density gradient centrifugation goes further in particle separation than differential centrifugation. It is ideal when the goal is to isolate particles of similar sizes, but different densities. In this case, it is possible to establish density gradient solutions with increasing concentrations of specific materials, in the spinning tubes (Figure 2). Cesium salt gradients are used in the separation of DNA, and sucrose gradients are used in subcellular fractionation to isolate organelles and multiprotein complexes, like ribosomes (Dumetre & Darde, 2004; Perper, Zee, & Mickelson, 1968). Today, there are several commercial gradient solution kits to isolate specific particles. Roughly, there are two types of density gradient centrifugation: rote-zonal centrifugation and isopycnic centrifugation (also called equilibrium centrifugation), which differ in the way particles are separated across the gradient (see Figure 2).

Figure 2: Types of preparative centrifugation. A – Rote-zonal centrifugation B – Isopycnic centrifugation (image credit: (Frei))

Rote-zonal centrifugation – particle separation depends mostly on particle mass. Zones, or bands, are generated, each containing a particle fraction of a specific mass. However, care must be taken when performing rote-zonal centrifugation. Because the mass of the particles is higher than the density of the solvent, if they are centrifuged for too long, all particles will eventually deposit in the bottom of the tube (Frei).

Isopycnic (equilibrium) centrifugation – particle separation depends solely on their density. In isopycnic separation, particles are mixed with the gradient solution, and during centrifugation, they will move until they reach the gradient phase which equals their density (isopycnic or equilibrium point). Because the density of the gradient medium is always higher than the density of particles, these will never sediment, independently of the centrifugation time. Continuous gradients may be used in isopycnic centrifugation, however, discontinuous gradients in which particles form bands at the interface between the density gradient layers are more suitable for the separation of some biological samples, like the separation of lymphocytes from whole blood (Frei; Ohlendieck & Harding, 2017).

Applications of Analytical and Preparative Ultracentrifugation

Due to their intrinsic differences, analytical and preparative ultracentrifugation are used for different purposes:

Analytical ultracentrifugation

  • determination of the purity (including the presence of aggregates) and oligomeric state of macromolecules, by recording sedimentation velocity data
  • determination of the average molecular mass of solutes in their native state
  • Study of changes in the molecular mass of supramolecular complexes,
  • using either sedimentation velocity, sedimentation equilibrium (or both)
  • the detection of conformation and conformational changes

Preparative ultracentrifugation

  • subcellular fractionation
  • affinity purification of membrane vesicles
  • separation of DNA components
  • colloid separation
  • virus purification
The Ultracentrifuge: How to Use and How to Care

Modern ultracentrifuges are heavy, sturdy equipment that requires certain know-how for proper usage and care.

  1. Rotor balance. As in all centrifuges, sample spinning requires a proper balance of the weight inside the rotor. Given the extremely high spinning speed inside the ultracentrifuge’s rotor, the impact of subtle imbalances may be shockingly strong. Modern ultracentrifuges have some buffer capacity, to absorb slight weight imbalances, and when there is too much imbalance, an automatic system shuts off the device. Moreover, in all ultracentrifuges, the rotor is encapsulated in a strong heavy metallic cage, to avoid vibrations and projections that could damage the sample and endanger the operator. Yet, it is of vital importance that the ultracentrifuge is properly loaded, according to the manufacturer’s instructions.
  2. Sample position in the rotor. All rotor positions must be filled. Even when there are only a few tubes, the rest of the positions must be occupied with blank samples of equivalent weight. To avoid both rotor and sample damage, it is important to set the ultracentrifuge to slow acceleration and deceleration modes. This is especially important in density gradients, as the sudden stop of the spinning may affect the separation of the gradient layers (Ohlendieck & Harding, 2017). Ultracentrifuges are expensive devices, which are required to accurately separate particles in solution. To ensure the proper function of the ultracentrifuge, care measures must be undertaken regularly. Apart from safety, proper loading of the rotor avoids excessive vibration, which can cause damage to the device.
  3. Centrifuge cleaning. Maintenance and cleaning of the rotor must be done with non-abrasive detergents to avoid corrosion. Rotor cleaning is especially important to ensure that there are no remnants of the samples that were centrifuged, and therefore, should always be performed after spinning.
  4. Storage. Whenever the device is not used, or simply for overnight storage, rotors must be kept in a dry room, properly cleaned, and left to dry in an inverted position, to avoid the accumulation of water in the sample cells.
  5. Regular maintenance. This should be done by certified operators to ensure the proper long-term function of the ultracentrifuge.
Advantages and Limitations of Ultracentrifugation

From the development of the first ultracentrifuge in the 1920s by Svedberg, up to today, the scientific advances that resulted from the application of ultracentrifugation to biology, chemistry, material science, and others, are countless. In its most obvious approaches, ultracentrifugation extended the limits of biology research to the subcellular level, by allowing the isolation of particles as small as ribosomes, subcellular organelles, membranes, and ribonucleic acids. With the advent of analytical ultracentrifugation, research took another step further towards the understanding of the submicroscopic world, with the ability to further characterize molecular size, shape, and structure. However, ultracentrifugation has its own limitations, like any other laboratory technique. These include:

  1. Low sample yield – In preparative ultracentrifugation, samples must be washed several times after spinning, to ensure that there is no cross-contamination between fractions. Samples for preparative centrifugation are usually limited in size (e.g., tissues) or volume (e.g., cell suspensions or blood). In every wash step that a sample is subjected to, there is a loss of material, and thus, after an ultracentrifugation protocol, the yield can be very low.
  2. Ultracentrifugation is still a time-consuming process, and it can take up to several hours to fractionate all the components of a single mixture.
  3. Ultracentrifuges are extremely expensive devices, which require constant maintenance, therefore ultracentrifuges are not routinely found in labs, but there is usually only one device per department or university.
Conclusion

To date, ultracentrifugation, in its most varied forms and protocols is used routinely and continues to be a fundamental approach not only in academic research but also in industry and the medical context. Nevertheless, it is fundamental to keep in mind how to properly use ultracentrifuges, to keep the sample and device user safe, and to ensure long-term functionality of the devices.

References
  1. (2019a). Laboratory Centrifuges. Retrieved November, 2019, from https://www.biocompare.com/Lab-Equipment/Laboratory-Centrifuges/
  2. (2019b). Ultracentrifuges. from https://www.biocompare.com/Lab-Equipment/10155-Benchtop-Ultracentrifuge/
  3. Cole, J. L. (2009). Analytical ultracentrifugation. In I. D. Wilson, E. R. Adlard, M. Cooke & C. F. Pole (Eds.), Handbook of methods and instrumentation in separation science (Vol. 1, pp. 34-41). Academic Press.
  4. Dumetre, A., & Darde, M. L. (2004). Purification of Toxoplasma gondii oocysts by cesium chloride gradient. J Microbiol Methods, 56(3), 427-430. doi: 10.1016/j.mimet.2003.11.020
  5. Frei, M. Centrifugation Separations. Retrieved December, 2019, from https://sigmaaldrich.com/technical-documents/articles/biofiles/centrifugation-separations.html#ref
  6. Jasinski, D. L., Schwartz, C. T., Haque, F., & Guo, P. (2015). Large scale purification of RNA nanoparticles by preparative ultracentrifugation. Methods Mol Biol, 1297, 67-82. doi: 10.1007/978-1-4939-2562-9_5
  7. Kumar, P. Methods used for Separation of Particles in Centrifugation: 3 Methods. Retrieved December, 2019, from http://www.biologydiscussion.com/biochemistry/centrifugation/methods-used-for-separation-of-particles-in-centrifugation-3-methods/12453
  8. Long, D. G., Borsa, J., & Sargent, M. D. (1976). A potential artifact generated by pelleting viral particles during preparative ultracentrifugation. Biochim Biophys Acta, 451(2), 639-642. doi: 10.1016/0304-4165(76)90162-8
  9. Martin, S. S., Giugliano, R. P., Murphy, S. A., Wasserman, S. M., Stein, E. A., Ceska, R., . . . Sabatine, M. S. (2018). Comparison of Low-Density Lipoprotein Cholesterol Assessment by Martin/Hopkins Estimation, Friedewald Estimation, and Preparative Ultracentrifugation: Insights From the FOURIER Trial. JAMA Cardiol, 3(8), 749-753. doi: 10.1001/jamacardio.2018.1533
  10. Momen-Heravi, F. (2017). Isolation of Extracellular Vesicles by Ultracentrifugation. Methods Mol Biol, 1660, 25-32. doi: 10.1007/978-1-4939-7253-1_3
  11. Ohlendieck, K., & Harding, S. E. (2017). Centrifugation and Ultracentrifugation.
  12. Perper, R. J., Zee, T. W., & Mickelson, M. M. (1968). Purification of lymphocytes and platelets by gradient centrifugation. J Lab Clin Med, 72(5), 842-848.
  13. Wasan, K. M., Cassidy, S. M., Kennedy, A. L., & Peteherych, K. D. (2001). Lipoprotein isolation and analysis from serum by preparative ultracentrifugation. Methods Mol Med, 52, 27-35. doi: 10.1385/1-59259-073-X:27

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Introduction

In behavioral neuroscience, the Open Field Test (OFT) remains one of the most widely used assays to evaluate rodent models of affect, cognition, and motivation. It provides a non-invasive framework for examining how animals respond to novelty, stress, and pharmacological or environmental manipulations. Among the test’s core metrics, the percentage of time spent in the center zone offers a uniquely normalized and sensitive measure of an animal’s emotional reactivity and willingness to engage with a potentially risky environment.

This metric is calculated as the proportion of time spent in the central area of the arena—typically the inner 25%—relative to the entire session duration. By normalizing this value, researchers gain a behaviorally informative variable that is resilient to fluctuations in session length or overall movement levels. This makes it especially valuable in comparative analyses, longitudinal monitoring, and cross-model validation.

Unlike raw center duration, which can be affected by trial design inconsistencies, the percentage-based measure enables clearer comparisons across animals, treatments, and conditions. It plays a key role in identifying trait anxiety, avoidance behavior, risk-taking tendencies, and environmental adaptation, making it indispensable in both basic and translational research contexts.

Whereas simple center duration provides absolute time, the percentage-based metric introduces greater interpretability and reproducibility, especially when comparing different animal models, treatment conditions, or experimental setups. It is particularly effective for quantifying avoidance behaviors, risk assessment strategies, and trait anxiety profiles in both acute and longitudinal designs.

What Does Percentage of Time in the Centre Measure?

This metric reflects the relative amount of time an animal chooses to spend in the open, exposed portion of the arena—typically defined as the inner 25% of a square or circular enclosure. Because rodents innately prefer the periphery (thigmotaxis), time in the center is inversely associated with anxiety-like behavior. As such, this percentage is considered a sensitive, normalized index of:

  • Exploratory drive vs. risk aversion: High center time reflects an animal’s willingness to engage with uncertain or exposed environments, often indicative of lower anxiety and a stronger intrinsic drive to explore. These animals are more likely to exhibit flexible, information-gathering behaviors. On the other hand, animals that spend little time in the center display a strong bias toward the safety of the perimeter, indicative of a defensive behavioral state or trait-level risk aversion. This dichotomy helps distinguish adaptive exploration from fear-driven avoidance.

  • Emotional reactivity: Fluctuations in center time percentage serve as a sensitive behavioral proxy for changes in emotional state. In stress-prone or trauma-exposed animals, decreased center engagement may reflect hypervigilance or fear generalization, while a sudden increase might indicate emotional blunting or impaired threat appraisal. The metric is also responsive to acute stressors, environmental perturbations, or pharmacological interventions that impact affective regulation.

  • Behavioral confidence and adaptation: Repeated exposure to the same environment typically leads to reduced novelty-induced anxiety and increased behavioral flexibility. A rising trend in center time percentage across trials suggests successful habituation, reduced threat perception, and greater confidence in navigating open spaces. Conversely, a stable or declining trend may indicate behavioral rigidity or chronic stress effects.

  • Pharmacological or genetic modulation: The percentage of time in the center is widely used to evaluate the effects of pharmacological treatments and genetic modifications that influence anxiety-related circuits. Anxiolytic agents—including benzodiazepines, SSRIs, and cannabinoid agonists—reliably increase center occupancy, providing a robust behavioral endpoint in preclinical drug trials. Similarly, genetic models targeting serotonin receptors, GABAergic tone, or HPA axis function often show distinct patterns of center preference, offering translational insights into psychiatric vulnerability and resilience.

Critically, because this metric is normalized by session duration, it accommodates variability in activity levels or testing conditions. This makes it especially suitable for comparing across individuals, treatment groups, or timepoints in longitudinal studies.

A high percentage of center time indicates reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance. reduced anxiety, increased novelty-seeking, or pharmacological modulation (e.g., anxiolysis). Conversely, a low percentage suggests emotional inhibition, behavioral avoidance, or contextual hypervigilance.

Behavioral Significance and Neuroscientific Context

1. Emotional State and Trait Anxiety

The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity, where animals exhibit persistent avoidance of the center due to heightened emotional reactivity. This metric can also distinguish between acute anxiety responses and enduring trait anxiety, especially in longitudinal or developmental studies. Its normalized nature makes it ideal for comparing across cohorts with variable locomotor profiles, helping researchers detect true affective changes rather than activity-based confounds.

2. Exploration Strategies and Cognitive Engagement

Rodents that spend more time in the center zone typically exhibit broader and more flexible exploration strategies. This behavior reflects not only reduced anxiety but also cognitive engagement and environmental curiosity. High center percentage is associated with robust spatial learning, attentional scanning, and memory encoding functions, supported by coordinated activation in the prefrontal cortex, hippocampus, and basal forebrain. In contrast, reduced center engagement may signal spatial rigidity, attentional narrowing, or cognitive withdrawal, particularly in models of neurodegeneration or aging.

3. Pharmacological Responsiveness

The open field test remains one of the most widely accepted platforms for testing anxiolytic and psychotropic drugs. The percentage of center time reliably increases following administration of anxiolytic agents such as benzodiazepines, SSRIs, and GABA-A receptor agonists. This metric serves as a sensitive and reproducible endpoint in preclinical dose-finding studies, mechanistic pharmacology, and compound screening pipelines. It also aids in differentiating true anxiolytic effects from sedation or motor suppression by integrating with other behavioral parameters like distance traveled and entry count (Prut & Belzung, 2003).

4. Sex Differences and Hormonal Modulation

Sex-based differences in emotional regulation often manifest in open field behavior, with female rodents generally exhibiting higher variability in center zone metrics due to hormonal cycling. For example, estrogen has been shown to facilitate exploratory behavior and increase center occupancy, while progesterone and stress-induced corticosterone often reduce it. Studies involving gonadectomy, hormone replacement, or sex-specific genetic knockouts use this metric to quantify the impact of endocrine factors on anxiety and exploratory behavior. As such, it remains a vital tool for dissecting sex-dependent neurobehavioral dynamics.
 The percentage of center time is one of the most direct, unconditioned readouts of anxiety-like behavior in rodents. It is frequently reduced in models of PTSD, chronic stress, or early-life adversity. Because it is normalized, this metric is especially helpful for distinguishing between genuine avoidance and low general activity.

Methodological Considerations

  • Zone Definition: Accurately defining the center zone is critical for reliable and reproducible data. In most open field arenas, the center zone constitutes approximately 25% of the total area, centrally located and evenly distanced from the walls. Software-based segmentation tools enhance precision and ensure consistency across trials and experiments. Deviations in zone parameters—whether due to arena geometry or tracking inconsistencies—can result in skewed data, especially when calculating percentages.

     

  • Trial Duration: Trials typically last between 5 to 10 minutes. The percentage of time in the center must be normalized to total trial duration to maintain comparability across animals and experimental groups. Longer trials may lead to fatigue, boredom, or habituation effects that artificially reduce exploratory behavior, while overly short trials may not capture full behavioral repertoires or response to novel stimuli.

     

  • Handling and Habituation: Variability in pre-test handling can introduce confounds, particularly through stress-induced hypoactivity or hyperactivity. Standardized handling routines—including gentle, consistent human interaction in the days leading up to testing—reduce variability. Habituation to the testing room and apparatus prior to data collection helps animals engage in more representative exploratory behavior, minimizing novelty-induced freezing or erratic movement.

     

  • Tracking Accuracy: High-resolution tracking systems should be validated for accurate, real-time detection of full-body center entries and sustained occupancy. The system should distinguish between full zone occupancy and transient overlaps or partial body entries that do not reflect true exploratory behavior. Poor tracking fidelity or lag can produce significant measurement error in percentage calculations.

     

  • Environmental Control: Uniformity in environmental conditions is essential. Lighting should be evenly diffused to avoid shadow bias, and noise should be minimized to prevent stress-induced variability. The arena must be cleaned between trials using odor-neutral solutions to eliminate scent trails or pheromone cues that may affect zone preference. Any variation in these conditions can introduce systematic bias in center zone behavior. Use consistent definitions of the center zone (commonly 25% of total area) to allow valid comparisons. Software-based segmentation enhances spatial precision.

Interpretation with Complementary Metrics

Temporal Dynamics of Center Occupancy

Evaluating how center time evolves across the duration of a session—divided into early, middle, and late thirds—provides insight into behavioral transitions and adaptive responses. Animals may begin by avoiding the center, only to gradually increase center time as they habituate to the environment. Conversely, persistently low center time across the session can signal prolonged anxiety, fear generalization, or a trait-like avoidance phenotype.

Cross-Paradigm Correlation

To validate the significance of center time percentage, it should be examined alongside results from other anxiety-related tests such as the Elevated Plus Maze, Light-Dark Box, or Novelty Suppressed Feeding. Concordance across paradigms supports the reliability of center time as a trait marker, while discordance may indicate task-specific reactivity or behavioral dissociation.

Behavioral Microstructure Analysis

When paired with high-resolution scoring of behavioral events such as rearing, grooming, defecation, or immobility, center time offers a richer view of the animal’s internal state. For example, an animal that spends substantial time in the center while grooming may be coping with mild stress, while another that remains immobile in the periphery may be experiencing more severe anxiety. Microstructure analysis aids in decoding the complexity behind spatial behavior.

Inter-individual Variability and Subgroup Classification

Animals naturally vary in their exploratory style. By analyzing percentage of center time across subjects, researchers can identify behavioral subgroups—such as consistently bold individuals who frequently explore the center versus cautious animals that remain along the periphery. These classifications can be used to examine predictors of drug response, resilience to stress, or vulnerability to neuropsychiatric disorders.

Machine Learning-Based Behavioral Clustering

In studies with large cohorts or multiple behavioral variables, machine learning techniques such as hierarchical clustering or principal component analysis can incorporate center time percentage to discover novel phenotypic groupings. These data-driven approaches help uncover latent dimensions of behavior that may not be visible through univariate analyses alone.

Total Distance Traveled

Total locomotion helps contextualize center time. Low percentage values in animals with minimal movement may reflect sedation or fatigue, while similar values in high-mobility subjects suggest deliberate avoidance. This metric helps distinguish emotional versus motor causes of low center engagement.

Number of Center Entries

This measure indicates how often the animal initiates exploration of the center zone. When combined with percentage of time, it differentiates between frequent but brief visits (indicative of anxiety or impulsivity) versus fewer but sustained center engagements (suggesting comfort and behavioral confidence).

Latency to First Center Entry

The delay before the first center entry reflects initial threat appraisal. Longer latencies may be associated with heightened fear or low motivation, while shorter latencies are typically linked to exploratory drive or low anxiety.

Thigmotaxis Time

Time spent hugging the walls offers a spatial counterbalance to center metrics. High thigmotaxis and low center time jointly support an interpretation of strong avoidance behavior. This inverse relationship helps triangulate affective and motivational states.

Applications in Translational Research

  • Drug Discovery: The percentage of center time is a key behavioral endpoint in the development and screening of anxiolytic, antidepressant, and antipsychotic medications. Its sensitivity to pharmacological modulation makes it particularly valuable in dose-response assessments and in distinguishing therapeutic effects from sedative or locomotor confounds. Repeated trials can also help assess drug tolerance and chronic efficacy over time.
  • Genetic and Neurodevelopmental Modeling: In transgenic and knockout models, altered center percentage provides a behavioral signature of neurodevelopmental abnormalities. This is particularly relevant in the study of autism spectrum disorders, ADHD, fragile X syndrome, and schizophrenia, where subjects often exhibit heightened anxiety, reduced flexibility, or altered environmental engagement.
  • Hormonal and Sex-Based Research: The metric is highly responsive to hormonal fluctuations, including estrous cycle phases, gonadectomy, and hormone replacement therapies. It supports investigations into sex differences in stress reactivity and the behavioral consequences of endocrine disorders or interventions.
  • Environmental Enrichment and Deprivation: Housing conditions significantly influence anxiety-like behavior and exploratory motivation. Animals raised in enriched environments typically show increased center time, indicative of reduced stress and greater behavioral plasticity. Conversely, socially isolated or stimulus-deprived animals often show strong center avoidance.
  • Behavioral Biomarker Development: As a robust and reproducible readout, center time percentage can serve as a behavioral biomarker in longitudinal and interventional studies. It is increasingly used to identify early signs of affective dysregulation or to track the efficacy of neuromodulatory treatments such as optogenetics, chemogenetics, or deep brain stimulation.
  • Personalized Preclinical Models: This measure supports behavioral stratification, allowing researchers to identify high-anxiety or low-anxiety phenotypes before treatment. This enables within-group comparisons and enhances statistical power by accounting for pre-existing behavioral variation. Used to screen anxiolytic agents and distinguish between compounds with sedative vs. anxiolytic profiles.

Enhancing Research Outcomes with Percentage-Based Analysis

By expressing center zone activity as a proportion of total trial time, researchers gain a metric that is resistant to session variability and more readily comparable across time, treatment, and model conditions. This normalized measure enhances reproducibility and statistical power, particularly in multi-cohort or cross-laboratory designs.

For experimental designs aimed at assessing anxiety, exploratory strategy, or affective state, the percentage of time spent in the center offers one of the most robust and interpretable measures available in the Open Field Test.

Explore high-resolution tracking solutions and open field platforms at
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References

  • Prut, L., & Belzung, C. (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. European Journal of Pharmacology, 463(1–3), 3–33.
  • Seibenhener, M. L., & Wooten, M. C. (2015). Use of the open field maze to measure locomotor and anxiety-like behavior in mice. Journal of Visualized Experiments, (96), e52434.
  • Crawley, J. N. (2007). What’s Wrong With My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. Wiley-Liss.
  • Carola, V., D’Olimpio, F., Brunamonti, E., Mangia, F., & Renzi, P. (2002). Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behavior in inbred mice. Behavioral Brain Research, 134(1–2), 49–57.

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