Zone Preference in the Visual Water Maze
June 26, 2025
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Polyacrylamide gel electrophoresis is one of the first forms of gel electrophoresis. Polyacrylamide is a synthetic polymer that is considered the most all-around separation matrix to date due to its transparency, electrical neutrality, and adjustable pore size. The technique can be applied to the analysis of proteins and nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Polyacrylamide gel electrophoresis (PAGE) is a form of gel electrophoresis that uses polyacrylamide gel as a support or a separation matrix. Similar to other forms of electrophoresis, polyacrylamide gels in PAGE stabilize the pH in the system and prevent the convection current that is induced during electrophoretic separation.
The shape and size of the space in-between polyacrylamide molecules also exert the molecular sieving influence, which imposes a frictional force on the components in the sample.[1,2] The pore size can be adjusted to meet the expected size range of the resolved components.
The gel is submerged in the electrophoresis running buffer, and samples in the liquid phase are mixed with a sample loading buffer containing a tracking dye and applied to the sample application well on the top of the gel.[1,3]
Electrophoretic separation begins when an electric field is applied to the unit. The components in the sample are gradually separated into distinct bands based on their characteristics, and the migration continues until the electric field is removed.
Larger components will migrate at a slower rate than smaller ones because they are inflicted with greater frictional force. The result of the separation can be visualized by staining the gel with dyes that are compatible to the gels and the samples.[4,5]
Polyacrylamide is a synthetic polymer that results from the polymerization of acrylamide and N,N’-methylenebisacrylamide, also referred to as ‘bis-acrylamide’. Both acrylamide and bis-acrylamide in their monomeric forms are extremely toxic, partly due to their aqueous forms, which make them easily absorbed by the skin.[6,7]
The polymerization is catalyzed by ammonium persulfate or potassium persulfate and by N,N,N’,N’-tetramethylethylenediamine (TEMED).[6] The addition of persulfate and TEMED accelerates the polymerization by increasing the formation of persulfate free radicals that convert acrylamide monomers to free radicals.
Acrylamide free radicals react with unactivated monomers, which in turn, initiate the polymerization of acrylamide monomers in a head-to-tail fashion.[3,5] Bis-acrylamide is cross-linked between acrylamide molecules in the polymerizing chain, forming a transparent, charge-neutral, and inert polyacrylamide gel.[3,6,7]
Since polymerization involves the generation of free-radical, the presence of oxygen can interfere with the gelation process because it can absorb the generated free radicals.
To circumvent such interference, polyacrylamide solutions are usually placed under a vacuum condition to degass, which will remove air dissolved in the solutions before the polymerization is initiated.[5]
Another popular approach to reducing exposure to oxygen is to let the polymerization occurs in a vertically-placed sealed cylinder tube or the space between two glass plates to create a slab gel.[5,7]
Alternatively, ammonium persulfate and TEMED can be replaced with riboflavin or riboflavin-5’-phosphate to catalyze the polymerization, in a process called photopolymerization.
In the presence of light and oxygen, riboflavin degrades and generates free radicals that react with acrylamide monomers. In the case of photopolymerization, the gelation process usually takes longer than the typical polyacrylamide polymerization.[3,5]
Polyacrylamide gels’ pore size is determined by the concentration of acrylamide and its cross-linking agent. The ratio between acrylamide and its cross-linker, bis-acrylamide is usually adjusted to obtain the desired sieving effect on the components being separated.[3,5,7]
The relationship between the pore size of acrylamide gels, the concentration of total acrylamide and bis-acrylamide can be expressed as a percentage of total acrylamide concentration (T), and the degree of cross-linking (C) as follows:
T = (a + b)/V x 100[%],
C = b/(a + b) x 100[%]
where T is the total acrylamide concentration,
C is the degree of cross-linking
a is the mass of acrylamide in g,
b is the mass of bis-acrylamide in g, and
V is the total volume of polyacrylamide solution in mL.
As a rule, the pore size increases as the T value increases and C remains constant. When T remains constant, however, the pore size is largest at high and low C values.[7]
The C-value of polyacrylamide gels is typically 3% for the electrophoretic separation of proteins and 5% for that of nucleic acids, which are translated to 29:1 acrylamide/bis-acrylamide and 19:1 acrylamide/bis-acrylamide, respectively.[3,6,7]
Higher percentage of C-value causes the gels to be brittle, opaque and relatively hydrophobic, making them difficult to handle and of little use in electrophoretic separation.[6,7]
In general, PAGE is performed using gels containing 3-30% acrylamide. Gels with lower acrylamide content (3-7.5%) are often used in the electrophoretic separation of nucleic acids, while those made with higher acrylamide content (8-30%) are used in the electrophoresis of proteins.[5]
Other than the pore size of the gel, PAGE can be modified by changing the setup and adjusting the gel composition to accommodate the objectives of the analysis. Since a polymerization reaction must be initiated for polyacrylamide gels, all necessary components must be present in a suitable proportion and condition.
This is to ensure that polymerization can occur without any inhibition and that the gels possess the desired characteristics. The following are examples of alterations that could affect the property of acrylamide gels and the subsequent PAGE:
Similar to agarose gel electrophoresis, PAGE can be modified to separate components in their denatured state by adding additives to the gels. Denaturants such as sodium dodecyl sulfate (SDS) and Triton X-100 can be added to polyacrylamide solution without affecting the gel’s property besides its denaturing state.
Other additives such as urea and formamide, however, not only denature the molecules of the components being separated but also disrupt the hydrogen bonds of the acrylamide monomers, resulting in the change of the gel’s molecular sieving influence.
The pore size of urea or formamide-containing polyacrylamide gels is generally smaller than the usual polyacrylamide gels.[3]
Other than bis-acrylamide, other cross-linking agents can be replaced to alter the property of the polymerized acrylamide gels.
N,N′-Bis(acryloyl)cystamine (BAC), N’,N’-(1,2-dihydroxyethelyne)bisacrylamide (DHEBA) and ethylene diacrylate (EDA), for example, can substitute bis-acrylamide to cast reversible gels, which can be solubilized after electrophoresis to recover the separated components.[6]
Another example of an alternative crosslinker is piperazine diacrylamide (PDA), which enhances the resolution of the polyacrylamide, while reducing the background of the gel that occurs from silver staining.[3]
Similar to bis-acrylamide, several monomer species have been synthesized to replace acrylamide. Acryloylaminopropanol (APP) is an example of a safer alternative to acrylamide, which is known to be carcinogenic and highly toxic to the nervous system.[6]
The two catalysts in the polyacrylamide solution, ammonium persulfate and TEMED, initiate the polymerization of acrylamide monomers.
Their absence or insufficient concentration extends the duration of the polymerization, which, if too long, could inadvertently allow oxygen to dissolve in the polyacrylamide solution, leading to non-uniform pore size and mechanically weak gel.
In contrast, too high a concentration of either catalyst results in shorter polyacrylamide chains, lesser gel transparency, and gel elasticity. In some cases, the excess catalysts can react with the components in the samples, altering the characteristics of the components being electrophoresed.
As with all chemical reactions, the polyacrylamide gel is sensitive to the temperature at which the polymerization takes place and the length of time it occurs.
The temperature has a direct effect on the rate of polymerization, which also affects the quality of the gel. Higher temperatures can, on one hand, accelerate the polymerization reaction; on the other, it can result in the formation of shorter polyacrylamide chains that lead to brittle and opaque gels.
On the contrary, lower polymerization temperature delays the gelation, and the resulting gel will be opaque, inelastic, and porous.
Typically, a temperature of 23-25°C is considered optimum for acrylamide polymerization. At this temperature range, gelation should be noticeable within 15-30 minutes for typical acrylamide polymerization and within 30-60 minutes for photopolymerization.[3]
PAGE is considered the technique of choice for the separation of proteins by electrophoresis due to its flexibility in the modification in the setup.
In most cases, electrophoretic separation of proteins is resolved in a discontinuous buffer system, called disc electrophoresis.
In this system, protein samples are resolved in a polyacrylamide gel consisting of a lower acrylamide-concentrated stacking gel on the upper layer and a higher acrylamide-concentrated resolving gel.
The stacking gel is made from 5% acrylamide gel and contains sample application wells. The migration of the components in the stacking gel is primarily based on their net charge, and the components are “stacked” in the order of their mobility at the border between the stacking and the resolving gels.
In the resolving gel, the components encounter frictional force, which causes them to migrate based on both their size and net charge.[7]
Proteins can be resolved based on their mobility and molecular weight in their natural conformation using a native PAGE or in their denatured state based solely on their size using an SDS-PAGE.
SDS-PAGE is more widely used for the determination of the protein size because the SDS has masked the overall net charge of the proteins and unfolded them, rendering their shape and conformation irrelevant to their mobility during electrophoresis.[5,7]
Nonetheless, native PAGE is particularly popular for the analysis of membrane proteins and multiprotein complexes.
This is because membrane proteins are usually dissolved in detergent-containing buffers that can interfere with the SDS, and the identification of the components in multiprotein complexes is more informative in light of their functionality, which can be achieved using the native PAGE that takes into account their native conformation.[6-8]
Both PAGEs can be performed in either continuous or discontinuous buffer systems. Native PAGE and SDS-PAGE can be combined in two-dimensional gel electrophoresis (2D-PAGE) so that higher resolution and more information are acquired.
For example, proteins can be resolved based on their net charge using native PAGE in the first dimension, followed by SDS-PAGE in the second so that the resolved components are further separated based on their mass.[8]
Unlike proteins, PAGE is not the preferable technique in the separation of nucleic acids due to the toxicity of non-polymerized polyacrylamide gels and the complexity in the preparation, when compared with agarose gels.[1]
Nevertheless, the finer porosity of the polyacrylamide gels allows PAGE to complement agarose gel electrophoresis.[6] The followings are examples of the application of PAGE separation of nucleic acids that cannot be performed using agarose gel electrophoresis:
PAGE originally constitutes the last step in DNA sequencing based on Sanger and Maxam-Gilbert methods.
In DNA sequencing, several DNA fragments of many lengths, each terminated with different species of nucleobases and differed by one nucleotide, are separated in a denaturing polyacrylamide gel. The pattern of migration of the DNA fragments is analyzed from the bottom to the top to obtain the sequence information.
Nowadays, with the development of fluorescent tags and capillary electrophoresis that can automatically record the readouts, DNA sequencing is typically performed using automated sequencing.
In PAGE, the mobility of nucleic acids is not only influenced by the length of the sequence but also by the nucleobases in the sequence. Changes in the nucleotide sequence will lead to changes in the secondary structure of the double-stranded DNA.
Here, the amplified DNA fragment of interest is heated together with that of the wild-type, and the resulting heteroduplexes are separated on a native PAGE to unveil the mobility shift of the heteroduplexes, in comparison to the corresponding homoduplexes and the single-stranded DNA molecules.[7]
Overall, the characteristics of polyacrylamide gels have made PAGE one of the most versatile techniques in gel electrophoresis despite the complicated preparation and the toxicity of the gel components.
The adaptability of polyacrylamide allows it to complement agarose and other matrices, which have made PAGE one of the earliest forms of electrophoresis that is essential and still relevant in biotechnology.
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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