Zone Preference in the Visual Water Maze
June 26, 2025
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Biocatalysts refer to proteins that drive all non-spontaneous chemical reactions in any biological system.
Also known as enzymes, they are sensitive to temperature and pH, and act only on their specific reactants, unlike inorganic catalysts. However, their activities can be modulated and controlled. Since enzymes are also active in vitro and are organic in nature, they have been isolated, modified, and used in many industries.
A non-spontaneous chemical reaction can only happen when the reactant molecules have obtained a sufficient amount of energy, termed activation energy. The sooner the reactants acquire the activation energy, the faster they can turn into their unstable transition state and begin to transform into (a) product(s).[1]
Thus, chemical reactions possessing high activation energy will require a tremendous amount of energy for their reactants to transform into products. In such cases, extra heat (thermal energy) or pressure (kinetic energy) is often applied so that the expected product(s) are generated in time.
Rather than putting in more energy to meet the activation energy, catalysts can be added to certain chemical reactions so that their activation energy is lowered. At the initiation of a catalyzed reaction, catalysts spontaneously interact with molecules of the reactants and encourage their transformation into products.
Towards the end of the reaction, catalysts are released from the catalyst-product complex and are reused in the next round. When the reaction reaches the reactant-product equilibrium, the amount of the catalyst that remains in the system is not different from the start of the reaction.[2]
In other words, catalysts guide the reactants to take a detour to avoid the high hill of activation energy. The detour is typically more complex, but the time it takes to reach the destination is far shorter.
Catalysts exist in many forms and are either readily available in nature or man-made. They can be in the same phase as the reactant (homogeneous catalysis) or a different phase (heterogeneous catalysis).[2] They can exist as small molecules such as ions, radicals, metal atoms, organometallic compounds and complexes, or as large and complex molecules like proteins, metal crystals or porous solid surfaces.[2,3]
Biocatalysts refer to enzymes, which are proteins that catalyze any chemical reaction that takes place in a living cell of any organism. Similar to inorganic catalysts, enzymes expedite the rate of chemical reactions by reducing the activation energy but at a higher turnover rate.
Because enzymes are fundamentally proteins that function in living organisms, most of their activities are optimal in the aqueous phase, in a condition that resembles the organism’s natural state, which is sensitive to changes in temperature and pH.[4]
One of the most unique features of biocatalysts is the specificity in the initiation and inhibition of their catalytic activities. This unique feature stems from the three-dimensional structure of enzymes, which are built up from intramolecular interactions between the amino acid species that are assembled into proteins.
Most amino acids in enzymes contribute to their geometric form, while only a few serve as residues at the catalytic center.[5] Also known as the active site, the catalytic center is a small site on the enzyme where enzyme-reactant and enzyme-product interactions take place.[1,6]
The shape of the active site resembles a small pocket or a narrow channel, which allows only reactants, referred to as substrates, that are complementary to the active site to access. Thus, only the intended reactant(s) can interact with the functional residues at the active site to initiate the enzyme’s catalytic activity.[1,5,6]
Enzymes are said to be absolute specific when they recognize only a particular substrate and group-specific when they recognize molecules that have a specific functional group.[5]
Similar to the initiation of biocatalysis, enzymatic activity can be inhibited when a molecule with a similar conformation to the substrate competes with the substrate and successfully binds to the active site.[1]
Other than substrate specificity, the regulation of biocatalytic activity is another hallmark of biocatalysts. Again, this unique feature is also due to the structural dimension of proteins.
For example, certain enzymes may require interactions with a specific cofactor or coenzyme for their activity. When bound to a cofactor or coenzyme, the enzyme alters its structure into an active form so that catalytic activity can be achieved.
Along the same line, some enzymes can specifically bind to a small molecule, termed ligand, at their non-catalytic binding site, the allosteric site. Enzyme-ligand binding influences the enzyme conformation, which can result in catalytic enhancement if the ligand is an activator or the suppression of its catalytic activity if the ligand is an inhibitor.[6]
The followings compare the main differences between enzymes and synthetic catalysts:
| Biocatalysts | Inorganic Catalysts |
|---|---|
|
Natural enzymes/proteins |
Non-enzymatic catalysts, e.g. ions, metal atoms, or solid surface |
|
Sensitive to temperature and pH changes |
Are less sensitive to temperature and pH change |
|
Catalyze only when interacting with a specific reactant (substrate) |
Catalyze various reactants |
|
Can be specifically regulated |
Cannot be regulated |
Biocatalysts use one or more of the following mechanisms to catalyze chemical reactions:
Biocatalysts can facilitate enzymatic reactions when they form weak and transient bonds with their substrates.[6] The formation of an enzyme-substrate bond brings all substrates in the reaction into contact, especially if the reaction is composed of more than one.
The enzyme-substrate bond also arranges the substrates in the right order and in the orientation that they have the most reactive chirality.[1]
In acid-base catalysis, the functional group in the active site can act as catalytic acids or bases. As catalytic acids in a general acid catalyzed reaction, the enzyme’s catalytic group donates protons to the substrate, while in general base catalysis, the catalytic group receives protons from the substrate.
Both acid and base catalysis enhance the reactivity of the substrate functional group, stabilize the transition state and facilitate bond cleavage. Many enzymes contain many functional residues which act as acids at one site of the substrate and simultaneously as bases at another. Such acid-base catalysis is termed concerted acid-base catalysis.[1,6]
Also known as nucleophilic catalysis, covalent catalysis involves the formation of a transient covalent bond between the enzyme and its substrate. A nucleophilic group of the enzyme binds with an electrophilic group of the substrate, which results in an unstable enzyme-substrate complex that possesses an electrophilic catalytic center.
After the electrons are withdrawn from the substrate, it’s transformed into a product, and the covalent bond between the enzyme and product is eliminated.[1,6]
Metalloenzyme is a group of enzymes that predominantly use metal ion catalysis. They contain metal ion cofactors and are only active when they are bound to their cofactors.[1] The positive charges in metal ions provide them with the ability to behave similarly to acids.
Nevertheless, metal ion charges can be higher than +1 and do not shift the pH of the system regardless of their concentration.[1,6] These characteristics allow metal ions to enhance the nucleophilicity of the system when they act as Lewis acids and accept electrons from the substrate even at neutral or basic pH.
Another way that metal ions participate in catalyzed reactions is to shield substrates or stabilize intermediates that are negatively charged. The shielding and stabilization of negatively charged species adjust the orientation and minimize any repulsing force from the substrates or intermediates.
As a result, the activation energy of the reaction is reduced, and the transformation of substrates to products is favored, or vice versa.[6]
The restricted shape of the enzyme active binding site creates an enclosed setting, to which only its complementary substrates and other highly similar molecules can gain access.
In electrostatic catalyzed reactions, the binding of the enzyme to its substrate brings about charge distribution around the active site, which stabilizes the transition state or the intermediates of the reaction.[6]
In this type of catalysis, biocatalysts react more favorably to the transition state than their substrates or product(s). In essence, the enzymes have the highest affinity to the transition state, higher than compared to the substrates and products.
This drives the reaction towards an increase in the concentration of the transition state, which in turn enhances the rate of the catalyzed reaction.[1]
Biocatalysts are active outside of living cells, given that all reaction components are present, and the temperature and pH condition is suitable for their catalytic activities. Due to enzyme specificity and high turnover rate, they have been applied across many industries. For instances:
All things considered; enzymes are similar to typical catalysts in that they expedite chemical reactions by redirecting them to the path with lower activation energy.
However, like proteins, enzymes are three dimensional ‘natural’ catalysts, whose structure affords them selectivity and regulatory means, unlike any inorganic catalyst.
Enzymes as biocatalysts are highly discriminatory, in terms of their reactants and products, and can catalyze complex reactions with a high turnover rate, which make them highly applicable in many industries.
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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