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Southern Blotting Protocol

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Southern blotting is a hybridization technique used for deoxyribose nucleic acid (DNA). The method was named after Edward M. Southern, who developed the technique in the 1970s. The method involves the transfer of DNA fragments from an electrophoresis gel to a membrane causing immobilization of the DNA fragments and bands are produced. After immobilization, the DNA fragments could be subjected to hybridization analysis, enabling the bands with sequence similarity to a labeled probe. Oligonucleotides, similar to the target sequence are designed. These oligonucleotides are chemically synthesized, radiolabeled, and then used for the cloned DNA fragments. Sequences hybridizing with the hybridization probe are further analyzed to obtain the full-length sequence of the gene of interest. Southern blotting could be used for homology-based cloning by exploring the amino acid sequence of the protein product of the gene of interest. Furthermore, southern blotting is used to identify methylated sites in particular genes. In addition to its use for DNA analysis, the immunologists have long used the southern blotting for gene identification in somatic rearrangements and transgene studies.

Principle

Southern blotting is based on the principle of separation of DNA fragments by gel electrophoresis followed by the identification by labeled probe hybridization. The DNA fragments are separated based on their size and charge during electrophoresis. The technique involves the transfer of electrophoresed DNA from the electrophoresis gel to a nitrocellulose membrane. The nitrocellulose filter is sandwiched between the gel and the stack of paper towels which draws the transfer buffer from the gel through capillary action. The desired DNA is detected using a labeled probe complementary to the desired DNA.

 

Apparatus & Equipment

The agarose gel for southern blotting is mounted on a filter paper dipped in a reservoir containing transfer buffer. The hybridization membrane is sandwiched between the gel and paper towels which draw the transfer buffer through the gel by capillary action. The DNA molecules are run on the membrane. Nitrocellulose or nylon serves as the membrane material. After transfer, the DNA is fixed onto the membrane. Then, the membrane is placed in a solution of labeled RNA, single-stranded DNA, or oligodeoxynucleotide complementary to the blot transferred DNA bands to be detected.

 

Training Protocol

Southern blotting onto nylon or nitrocellulose membrane (Brown, 2001)

  1. Digest the DNA using the restriction endonuclease(s), run in an agarose gel with appropriate DNA size markers, dye the nucleotide strands with ethidium bromide, and identify the band position by visualizing the membrane.
  2. Treat the gel with distilled water ∼10 gel volumes 0.25 M HCl for 30 minutes, again with distilled water ∼10 volumes denaturation solution (1.5 M NaCl/0.5 M NaOH) twice for 20 minutes, and then with distilled water ∼10 volumes neutralization solution (1.5 M NaCl/0.5 M Tris-Cl, pH 7.0) for 20 minutes for twice, with gentle shaking at room temperature.
  3. Assemble the transfer stack consisting of a sponge, Whatman 3MM paper, the gel, pieces of plastic wrap, a nylon membrane equilibrated in distilled water or nitrocellulose membrane, five pieces of Whatman 3MM paper, and a 4-cm stack of paper towels. As each layer is applied, wet it with 20× saline sodium citrate (SCC) and remove air bubbles if any by carefully rolling a 10-ml glass pipet over the surface.
  4. Place a glass plate on top of the stack and put a 0.2 to 0.4-kg weight on the top to hold the stack in place. Leave it overnight.
  5. Recover the membrane. Mark the positions of the wells on the membrane and cut one corner to mark the orientation.
  6. Rinse the membrane in 2× saline sodium citrate (SSC), then place it on a sheet of Whatman 3MM paper and let it dry thoroughly.
  7. Wrap nylon membrane in UV-transparent plastic wrap and place DNA-side-down on a UV transilluminator (254-nm wavelength).

Note: The nitrocellulose membranes should be placed between two sheets of Whatman 3MM paper and baked under vacuum at 80°C for 2 hours.

 

Mutation detection by southern blotting (Mellars. & Gomez., 2011)

DNA restriction

  1. Dispense 10 mg of deoxyribose nucleic acid in a 500 ml microtube.
  2. Add 4 mL of 10× restriction enzyme buffer. Make the final volume to 40 mL by adding the distilled water.
  3. Add 1mL of Bcl1 restriction enzyme.
  4. Vortex the mixture, pulse-spin, and incubate it at 50°C overnight.

Gel preparation and DNA separation

  1. Assemble a 25 cm long gel casting tray and gel comb.
  2. Make 300 mL of 0.5% agarose (300 mL 1× Tris-borate ethylene diamine tri-acetic acid (TBE) and 1.5 g agarose).
  3. To dissolve the agarose by heating in a microwave or on a hot plate.
  4. Let it cool to 55–60°C and add 25 mL ethidium bromide.
  5. Pour it into the casting tray, remove air bubbles, and let it set.
  6. Prepare the samples after incubation and add 1.5 mL of 10% sodium dodecyl sulfate (SDS) and 8 mL of 6× sucrose loading buffer.
  7. Prepare the molecular weight marker by adding 0.5 mg–10 mL of distilled water and 5 mL of loading buffer.
  8. Remove the gel comb, place it in the electrophoresis tank, and cover it with 1× TBE.
  9. Carefully load the samples into the gel and run the samples on the gel from negative to positive at 45 V, for 40 hours.

 

Southern blotting

Gel preparation

  1. Carefully visualize the gel on the transilluminator. Spot the area of interest by noting the sizes of the bands of the marker.
  2. Carefully inject a small amount of ink into the position of two of the bands of the marker nearest to the area of interest. Cut the top and bottom of the gel and isolate the targeted section.
  3. Transfer it to a sandwich box and immerse in 0.25 M HCl for 10-15 minutes, on an orbital shaker for depurination.
  4. Replace the HCl with 500 mL denaturing buffer (1.5 M NaCl, 0.5 M NaOH) and leave the sandwich box on an orbital shaker for 60 minutes at low speed at room temperature.
  5. Replace the denaturing buffer with 500 mL neutralizing buffer (0.5 M Tris–HCl, 1.5 M NaCl. pH 7.3, adjust with 10 M NaOH). Leave the box on an orbital shaker at low speed for 90 minutes at room temperature.

Blot assembling

  1. Place the casting tray upside down in a large plastic tray to support the gel. Lay three to four layers of Whatman filter paper over the casting tray for the wick.
  2. Pour 500 mL of 20× SSC on the filter paper and saturate the base of the large with it.
  3. After 90 minutes in neutralizing buffer, slide the gel onto a casting tray and transfer it to the prepared blotting base.
  4. Surround the gel with cling film to cover the exposed area of Whatman filter paper up to the gel edge.
  5. Add 450 mL distilled water to 50 mL of 20x SSC to make 2× SSC.
  6. Cut a piece of Hybond N membrane slightly larger than the gel. Soak it in 2× SSC and then layer it onto the gel.

Note: Make sure to remove entrapped air bubbles if any.

  1. Cut four sheets of Whatman filter paper slightly larger than the gel. Soak them in 2× SSC and layer them onto the blot.
  2. Place a dry piece of Whatman filter paper on the blot and then place a stack of tissue approx. 6 cm in height covering the whole gel. Then, cover this with a stack of paper towels.
  3. Place a glass plate and a 500 g weight on the stack. Leave the blot overnight.
  4. Remove the paper towels, tissues, and filter paper the following morning.
  5. Rinse the Hybond membrane briefly in 2× SSC and blot dry using the filter paper.
  6. Mark the membrane with the help of a soft-lead pencil.
  7. Fix the DNA on the membrane by placing it under UV light for 5 minutes.

Probe labeling

  1. Add 30 ng of the designed probe to 5 mL of the primer mix and denature for 5 minutes at 99°C and then keep the mixture on ice.
  2. Add labeling buffer, containing the dNTPs, with 2.5 mL of 32P and 2 mL of Klenow fragment polymerase.
  3. Incubate the mixture to allow incorporation of the radiolabel for 10 minutes to 60 minutes at 37°C.

Hybridization

  1. Turn ON the hybridization oven 30 minutes before use and heat to 65°C.
  2. Warm 20 mL of 2× SSC in a water bath.
  3. Warm 10 mL of PerfectHyb Plus in a water bath.
  4. Roll the blot up with the DNA side inside and the pencil mark to the outside.
  5. Place the blot in a hybridization bottle and add 20 mL warmed 2× SSC.
  6. Unroll the blot on the bottle wall by tilting and rolling the bottle. Make sure no air bubble enters the blot.
  7. Pour the SSC and 10 mL of pre-warmed PerfectHyb Plus buffer.
  8. Tightly cap the bottle and place it in the oven.
  9. Pre-hybridize for 30 minutes up to a maximum of 4 hours.
  10. Denature the labeled probe by heating it to 100°C for 5 minutes and then placing it on ice.
  11. Warm 10 mL of PerfectHyb Plus buffer to 65°C.
  12. After pre-hybridization, remove the bottle from the oven and pour off the pre-hybridization buffer.
  13. Pour the probe solution in 10 mL of warmed buffer and immediately add to the blot.
  14. Replace the bottle in the oven and rotate at 65°C overnight.

Blot washing

  1. Pour 250 mL of washing buffer in a flask and warm to 65°C.
  2. Remove the bottle from the oven and discard the excess buffer.
  3. Detach the membrane with tweezers and place it in a plastic sandwich box.
  4. Add half of the buffer, seal the box, and place in a shaking water bath at 65°C for 15 minutes. Keep the remaining buffer at 65°C.
  5. Pour off the wash buffer and add the remaining. Wash for a further 15 minutes.
  6. Pour off the buffer and dry the blot using the Whatman filter paper.
  7. Run the Geiger counter over the blot.
  8. Air-dry the blot and wrap in cling film.

Autoradiography

  1. Tape the blot onto an X-ray film and pace another film on the top to make a sandwich autoradiograph in a darkroom.
  2. Place at -70°C for 48 hours before developing the first film.
  3. If bands are weak, replace the cassette in the freezer for 24-48 hours before the second film.
Applications

Characterization of human herpesvirus strains (Aubin. et al., 1991)

Southern blotting is a valuable technique for genome analysis and characterization. In the study, eight human herpesvirus 6 (HHV-6) strains were analyzed using the Southern blot and polymerase chain reaction. Nucleic acids from infected cells were digested by restriction enzymes and hybridized with cloned BamH1 fragments. In parallel, the DNA was amplified using the polymerase chain reaction employing primers derived from the strain SIE nucleotide sequence. The results indicated that all the strains were closely related to one another. However, marked differences in restriction patterns were observed in two groups, typified by strains SIE and HST, respectively. The findings of the study suggest that the HHV-6 species exhibit genetic polymorphism, and the sequences could be used as useful markers for the diagnosis and molecular epidemiology of HHV-6 infections.

Tracking of mouse cell lineage (Lo., Coulling., & Kirby., 1987)

The analysis of cell lineage has provided detailed insights into the underlying mechanisms of pattern formation, determination, and differentiation of cells in developmental systems. The feasibility of DNA microinjection for mouse embryos lineage studies was determined. For this, tissues from three transgenic mice mosaic with an exogenously introduced mouse β-major globin gene were analyzed using the Southern-blotting and in situ hybridization. The results presented early segregation of cells in the somatic and germ cell lineages. The in-situ hybridization data suggested that cells of the transformed lineages are finely dispersed which indicates an extensive cell-cell mixing during mouse development. Whereas the kidney cells had a patchy distribution, the transformed cells in the villi were aggregated, and the positive and negative seminiferous tubules were segregated in the testis. The study suggested a clonal basis for the organ development stages for kidney, intestine, and testis. The Southern blotting and in-situ hybridization allowed the direct quantification and localization of lineage descendants derived from the founder cells (marked or transformed).

Analysis of Epstein-Barr viral genomes in lymphoid malignancy (Ohshima. et al., 1990)

Southern blot analysis was used for the analysis of Epstein-Barr virus (EBV) genome and its comparison with non-neoplastic lymphadenopathic and healthy thymus tissues. In specimens marked positive by the Southern blot and PCR, in-situ hybridization studies were performed. By Southern blot analysis, it was found that 26% Hodgkin’s disease samples, 5.6% B-cell lymphomas (5.6%) and 11% T-cell lymphomas had EBV DNA. While in some samples, EBV DNA was not detected. The results indicate that the presence of EBV DNA is not related to lymphoid malignancy; however, the enhancement of the DNA could lead to neoplastic conditions.

 

Precautions
  • Wear gloves and a lab coat while handling the materials for Southern blotting.
  • Make sure no air bubbles got entrapped in gel, membrane, and Whatman filter paper as they may cause the DNA to run irregularly.
  • Monitor the agarose while heating, as it is liable to boil quickly.
  • Gently remove the gel comb to avoid well collapse.
  • Slowly release the sample in the wells for a smooth transfer.
  • Once the nitrocellulose membrane is placed, do not move it as the DNA transfer may already have taken place.

 

Strengths and Limitations
  • Southern blotting is a suitable technique to analyze long stretches of DNA such as the multikilobase restriction fragments.
  • The expression levels and expression profiles are better detected as compared to the other genomic analyses methods.
  • Southern blotting has a wide array of applications in the fields of DNA fingerprinting, including paternity and maternity testing, forensic studies, and personal identification.
  • The method is cheaper than genome sequencing and is useful to quantify the amount of DNA as well.
  • The method may require a large quantity of high-quality DNA as compared to other genome analyzing techniques.

 

References
  1. Brown, T. (2001). Southern blotting. Curr Protoc Immunol, Chapter 10:Unit 10.
  2. Mellars., & Gomez., k. (2011). Mutation detection by Southern blotting. Methods Mol Biol, 688, 281-291.
  3. Ohshima., M. Kikuchi., F. Eguchi., Y. Masuda., Y. Sumiyoshi., H. Mohtai., . . . Kimura., N. (1990). Analysis of Epstein-Barr viral genomes in lymphoid malignancy using Southern blotting, polymerase chain reaction and in situ hybridization. Virchows Arch B Cell Pathol Incl Mol Pathol, 59(6), 383-90.
  4. J. Aubin., H. Collandre., D. Candotti., D. Ingrand., C. Rouzioux., M. Burgard., . . . Agut., H. (1991). Several groups among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction. J Clin Microbiol, 29(2), 367-72.
  5. C. Lo., M. Coulling., & Kirby., C. (1987). Tracking of mouse cell lineage using microinjected DNA sequences: analyses using genomic Southern blotting and tissue-section in situ hybridizations. Differentiation, 35(1), 37-44.

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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.

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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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