Zone Preference in the Visual Water Maze
June 26, 2025
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Clark Oxygen Electrode is a silver-platinum electrochemical cell whose electrodes are covered by non-conductive membranes. They are used to measure gaseous or solubilized oxygen in the liquid.
Clark oxygen electrodes rely on the limited permeability to oxygen of non-conductive membranes, which reduces oxygen loss at the electrodes and allows the quantification in circulating blood.
The Clark electrodes are one of the inventions that become the basis of modern blood gas analyzers, which quantify the blood pH, oxygen, and carbon dioxide level in vitro and in vivo.
The same principle used for Clark oxygen electrodes has been adapted to build other gas measuring devices and biochemical sensors for medical, agricultural, and other industrial applications.
Clark electrodes directly quantify ions and/or molecules in their surrounding solution using polarography.[1]
Two electrodes, called the anode and cathode, are immersed in a chloride solution, or electrolyte. When an electric current is applied, electrochemical reactions take place on the surface of either electrode, altering the electrical characteristics of the components in the solution between the two electrodes.
Changes in electrical current in the component of interest can be measured and compared with known standards, enabling the quantification of the ion or molecules of interest.[1-2]
The predecessor of the Clark electrodes consists of two electrodes made from silver (Ag) and bare platinum wire (Pt).
Both electrodes are immersed in saturated potassium chloride (KCl) solution. The silver electrode serves as the anode and reference electrode. The platinum electrode is naked and acts as the cathode, which is in direct contact with the fluid of interest, whose oxygen level is being measured.
When an electric current of known magnitude is applied to the electrode, the ions in the KCl solution become an electrolyte, forming chloride and other ions. Simultaneously, the following reactions take place at the surface of the electrodes:
At the anode:
4Ag + 4Cl– —> 4AgCl + 4e–
Here, the Ag-electrode is oxidized into silver ions. Subsequently, silver ions interact with chloride ions from the electrolyte, forming silver chloride (AgCl). The resulting electrons participate in a reduction of oxygen at the Pt-cathode.
At the cathode:
O2 + 4H+ + 4e– —> 2H2O
Here, the Pt-electrode acts as a catalyst, reducing the dissolved oxygen in the sample to water. The reaction consumes electrons generated at the anode.
The combined reactions at either electrode can be expressed as:
4Ag + O2 + 4H+ + 4Cl– —> 4AgCl + 2H2O
The net reaction between both electrodes can be summarized into:
O2 + 4H+ + 4e– —> 2H2O
The electron flow between the electrodes generates a current, which is in direct proportion to the partial pressure of oxygen (pO2) dissolved in the sample. In medicine, this is referred to as the oxygen tension, which reflects the level of oxygen in the arterial blood.[2-3]
While the predecessor of Clark electrodes can quantify oxygen in the blood both in vitro and in vivo, there were some limitations in the use of naked platinum cathodes.
First and foremost, the electrochemical reactions consume high oxygen at the electrodes due to the unrestricted oxygen diffusion rate. Thus, the quantification of oxygen levels is often inaccurate and requires rapid continuous fluid flow to allow frequent contact of fresh samples with the electrodes.
Another major limitation is when Clark electrodes’ predecessor is used to measure oxygen in tissues of living organisms. Specifically, the naked platinum cathode does not restrict cellular proteins and other non-target components from participating in chemical reactions on its surface.
As a result, unintended reactions can alter the characteristics of these cellular components, which not only interferes with the measurement but can also poison the tissue or organism.[3-4]
Figure 1: The Clark Oxygen Electrode (Credit: “File: Clark Oxygen Electrode.svg” by Larry O’Connell is licensed under Creative Commons Attribution-Share Alike)
To address such limitations, Leland C. Clark Jr. used non-conductive membranes to shield the cathode and sealed both electrodes in an insulated glass tube.
The non-conductive membranes such as polyethylene, polytetrafluoroethylene (Teflon), and cellophane are semi-permeable. They only allow oxygen and other permeable molecules in the sample to pass through the membrane.
Consequently, the membrane serves as a barrier that bars non-target molecules from reaching the cathode. As a result, non-target reactions on the surface of the platinum cathode are eliminated, reducing the measurement errors and damage to living cells.
At the same time, the addition of non-conductive membranes also traps oxygen molecules at the cathode after they pass through the membrane. This way, oxygen diffusion is reduced, enhancing the accuracy of the measurement.[3-4]
The Clark electrode was first invented as a medical device that continuously monitors oxygen levels during medical procedures such as cardiovascular surgery.
Nowadays, the Clark oxygen electrode has been adapted to measure the oxygen level in other applications.
For instance:
However, the Clark electrode has also been modified or combined with other types of electrodes in many fluid-measuring devices.
For example:
In the glucose biosensor, the enzyme glucose oxidase (GOx) is trapped between two semi-permeable dialysis membranes. The first membrane next to the electrode is permeable to hydrogen peroxide (H2O2). The other membrane, which is permeable to glucose, is in direct contact with the sample.
The enzyme GOx catalyzes an oxidoreduction reaction, using glucose and oxygen as substrates. When glucose and oxygen diffuse from the sample and pass through the first dialysis membrane, GOx oxidizes glucose, transforming it into gluconic acid.
Simultaneously, oxygen is reduced to H2O2, which passes through the second dialysis membrane to the platinum electrode. At the surface of the platinum anode, H2O2 is oxidized to oxygen, which resupplies the GOx-catalyzed reaction.
At the same time, the electrons from H2O2-oxidation flow to the silver cathode, reducing it to silver ions.
The GOx-catalyzed oxidoreduction occurs continuously as long as glucose and oxygen are present. Ultimately, the level of glucose can be calculated from the number of oxygen consumed.
GOx-based enzyme electrode is regarded as the first biosensor. Since its conception, many biosensors have been developed to measure glucose levels and other components in body fluids.
Modern-day biosensors often use other oxidases and contain additional elements. Nevertheless, most of them are based on the same principle as the GOx-based enzyme electrode.[7]
Compared to its predecessor, the Clark electrode greatly improves the accuracy in oxygen quantification and is adaptable for the measurement of various biologically relevant fluids.
Nonetheless, the Clark electrode still possesses the following limitations:[8]
The Clark electrode is dependent on the presence of oxygen. In other words, the accuracy of the measurement using Clark electrode or biosensors modified from it relies on oxygen, which can be difficult in cases of samples with small volumes, large surface areas, or changing temperature.
In addition, oxygen can also interfere with the measurement, causing measurement or sensor errors. For example, gas bubbles in the electrolyte arising from leaking glass tubes can change the concentration of the fluid between the two electrodes.[8-9]
The Clark electrode relies on the interaction between the electrolytes and the electrodes. This implies that water is necessary for the redox reactions to initiate. As such, this electrode is only applicable to hydrophilic or amphiphilic fluids.
Since one of the electrodes must be in direct contact with the sample of interest, the Clark electrode-based medical equipment is typically invasive. That is, most Clark electrode-based medical devices will damage the tissue and require specialized training to ensure safe use.
All in all, the Clark electrode addresses the limitations of its predecessor by introducing non-conductive membranes to the cathode, which lessens the rate of oxygen diffusion.
Originally conceived as a medical device, the Clark electrodes have been adapted for non-medical uses. It is also the foundation of modern-day gas measuring devices and biosensors, which not only measures oxygen but also other biologically relevant molecules.
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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