Zone Preference in the Visual Water Maze
June 26, 2025
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Gel electrophoresis is a type of electrophoresis that separates molecules or components based on their size or conformation in a matrix made from gel-forming substances.
Since its conception, gel electrophoresis has been modified, in terms of gel types, composition, and the set-up of the system to accommodate several aspects of the characterization of biomolecules.
It is nowadays regarded as one of the fundamental analytical methods in biochemistry, molecular biology, and clinical pathology.
The original intent of using a gel as a support matrix is to overcome the limitations of earlier support matrices for zone electrophoresis.
In zone electrophoresis, liquid samples are individually mixed with a loading buffer and applied onto a restricted area or zone of the support matrix that is saturated with electrophoresis running buffer. To initiate electrophoresis, an electric field is applied and removed when the separation is completed.
Components in the sample with the same characteristics are segregated into distinct bands or zones on the support matrix. Afterwards, the matrix is stained for visualization and further quantitative analyses.[4,9]
Support matrices in zone electrophoresis act as anti-convective stabilizers that mitigate the heat generated during electrophoresis.
When the electric current is met with the electrical resistance from the ionized components of the running buffer, the support matrix stabilizes the pH of the buffer surrounding the sample. This, in turn, prevents convection current induction and allows electrophoretic separation to continue without any disruption.
As a result, distinctive components obtained from zone electrophoresis are disjointed and not overlapped with nearby components, unlike the result obtained from moving boundary electrophoresis.[4]
Despite its obvious advantages over moving boundary electrophoresis, zone electrophoresis did not become widespread until the introduction of gel as a support matrix. This is because early matrices possess certain limitations.
For example, a bed of moist starch grains provides a good resolution, but the process of non-sample protein identification is time-consuming and labor-intensive.[5]
Another matrix, the filter paper, fails to provide the resolving power that is on par with moving boundary electrophoresis. It is unsuitable for the analysis of biomolecules that exist only in a small fraction of a sample, nor can it accommodate the analysis of large-volume samples due to its absorbability.[4-6]
The introduction of gel made from potato starch overcomes the shortcomings of earlier support matrices. Contrary to filter paper, boiled starch has a low absorbability once it cools and gels.
Before the gel is fully set, the liquefied gel can be cast into a block or slab of various height, length, and thickness, enabling the modification of the sample application area to accommodate both small and large sample volumes. The process of non-sample protein identification is no longer necessary.[5]
In addition to the low absorbability, gel exerts a molecular sieving influence on the molecules of the components being electrophoresed.[5] The net charge of the molecules of the support matrix, the shape and size of the pores in-between impose additional friction onto the electrophoresed components.
Larger components will be imposed to greater friction and migrate slower than smaller components and vice versa.[3,4]
In other words, gel serves not only as an anti-convective support matrix but also as a separation matrix that sieves the components during electrophoresis run based on their masses, adding to the resolving power of the technique.
The gel’s lesser absorbability and its additional molecular sieving property improve the resolving power of zone electrophoresis to the extent that it is considered superior to that of moving boundary electrophoresis.[5]
Moreover, gel electrophoresis can be easily adapted to meet the desired resolving power and other objectives by fine-tuning the gel concentration and adjusting other compositions, making the technique highly versatile.[8]
Starch gels are the first gel used as a support matrix for zone electrophoresis. Starch gels were prepared using soluble potato starch at about the 10-16% (w/v) concentration in the electrophoresis running buffer.
The starch solution was boiled shortly and cast in a plastic tray, with slots or slits on the top for the sample application. After electrophoresis, the gel is stained with compatible dyes to visualize the pattern of separation.[5]
Nowadays, starch gels have been replaced by agarose and polyacrylamide gels, which possess less batch-to-batch variations than potato starch.[9]
Agarose is a natural linear polysaccharide isolated from red seaweed agar. It consists of alternating chains of 1,3-linked β-D-galactose and 1,4-linked 3, 6-anhydrogalactose.
Agarose gel is prepared similarly as a starch gel, using a casting tray containing a comb that molds the slots or wells for each sample. The concentration of agarose determines the resolution of the electrophoresis: the higher the concentration, the greater the resolving power.[9]
Agarose gel electrophoresis is a matrix of choice for the electrophoretic separation of linear nucleic acid fragments. It is generally suitable for the separation of DNA fragments ranging from 100 base pairs to 20 kilobase pairs.[7]
Larger DNA fragments are typically separated by pulsed-field gel electrophoresis, a modified version of gel electrophoresis.[8]
In addition to nucleic acids, agarose gel electrophoresis applies to electrophoretic separation of proteins.
A low concentration of agarose gel can be used for isoelectric focusing (IEF), a form of electrophoresis that separates amphoteric molecules based on their isoelectric point (pI).[8]
Recently, an agarose gel system combined with sodium dodecyl sulfate (SDS) has been developed to separate large proteins, ranging from 200-4,000 kDa.[2]
Polyacrylamide refers to a product of polymerization of acrylamide monomers in the presence of bisacrylamide (N,N-methylene-bisacrylamide), a crosslinking agent.
The reaction is catalyzed by ammonium persulphate (APS) and N,N,N’,N’-tetramethylenediamine (TEMED), and the concentration of polyacrylamide gel is dependent on a proportion of acrylamide monomer and the crosslinking agent, bisacrylamide.[3]
To cast a polyacrylamide gel, the polymerization solution is poured between a space of two vertically glass plates that are placed in parallel and sealed at the bottom, or a glass cassette.
For a vertical gel system, a comb is placed on top of the gel to create sample wells where the individual sample is applied and concentrated.
In a horizontal gel system, samples are typically applied onto the surface of a strip of filter paper or other materials, which are directly placed on top of the gel.[8-9]
Polyacrylamide gels are applicable for the electrophoretic separation of both nucleic acids and proteins.
For nucleic acids, agarose gels are preferable due to the toxicity of non-polymerized polyacrylamide gels, and the complexity in the preparation of polyacrylamide gels. Nonetheless, polyacrylamide gels can be modified to acquire superior resolution.[8]
Denaturing polyacrylamide gel, for example, is capable of distinguishing single-stranded DNA molecules that are only one nucleotide different, allowing the application to Maxam-Gilbert and Sanger sequencing.[9]
For proteins, polyacrylamide gels are the norm for electrophoretic separation because they are more adaptable to accommodate several aspects of protein characterization.
For example, proteins can be distinguished based on the differences in their natural structure using non-denaturing, or native polyacrylamide gels.[9]
Conversely, electrophoretic separation of proteins based on their size is achievable in higher percentage (10%-20%) polyacrylamide gels with sodium dodecyl sulfate (SDS), which denatures the proteins, allowing the proteins to migrate based on the size rather than the conformation.[8]
Gel electrophoresis is regarded as a highly adaptable technique and can be, first and foremost, refined by changing the type of gels, the gel concentration, and composition.
Additionally, gel electrophoresis can be further modified to meet the objectives of the analysis, as follows:
Additional information on the samples being analyzed can be obtained by performing additional electrophoresis in the direction that is perpendicular to the first round, or second-dimensional electrophoresis.
In the case of gel electrophoresis, the gel from the first round can be directly taken for the second-dimensional analysis, or it can be processed or modified beforehand.[10] This form of modification is applicable to both agarose and polyacrylamide gel electrophoresis.
While gel electrophoresis is initially performed in a homogenous buffer system as with the case of zone electrophoresis, it can be conducted in a non-homogenous or discontinuous buffer system.[9]
This is achieved by using different gel-casting and electrophoresis running buffers and by combining two gels made from different buffers. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the most well-known discontinuous gel electrophoresis.
In SDS-PAGE, an acrylamide gel consists of a stacking gel on the upper part and a separating gel on the lower part.
Stacking gels possess larger pores than separation gels and serve as a sample concentration site. Electrophoretic separation occurs in the separating gels, which possess smaller pores.[1]
Another modification of gel electrophoresis is the use of denaturants in the system. Denaturants or dissociating agents are added to gel-casting, sample loading, and running buffers to destabilize intramolecular bonds of nucleic acids and proteins.
Denaturing conditions force nucleic acids to exist as single-stranded molecules and proteins to unfold. As a result, the mobility of these denatured molecules during electrophoresis is solely based on their size and not their conformation.
In contrast to denaturing gels, nondenaturing or native gels allow for the analysis of nucleic acids and proteins based on their conformation, which can give functional information of the biomolecules under investigation.[9]
Gradient gels are polyacrylamide gels whose concentrations uniformly vary throughout the gel. This is achieved by pouring the higher concentrated gel to the bottom, followed by a mixture of the higher and lower concentrated gels.
Once the gel is completely polymerized, the concentration at the bottom of the gel is higher than that at the top. In other words, the sieving influence of gradient gels increases as the separating components migrate from the top to the bottom of the gel.[8]
The changing concentration of gradient gels creates a zone of sharpening effect, leading to higher resolving power than a single-percentage gel of the same range and allows for the electrophoretic separation of proteins with similar masses and the determination of the molecular diameter of proteins in their native states.[9]
Gel electrophoresis was initially conceived as another type of zone electrophoresis but has since been adapted to accommodate other types of electrophoretic separation.
In addition, gel types, their condition, composition and the electrophoresis system can be refined to meet the goals of the analysis being performed.
The resolving power of the technique, its simplicity and versatility have made the technique a staple of biochemistry and molecular biology research, as well as clinical diagnosis.
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.
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:
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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 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.
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).
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.
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.
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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