Zone Preference in the Visual Water Maze
June 26, 2025
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The spectrophotometer is a common optical device that measures the intensity of light relative to color (or wavelength). In the lab, they are used to determine how much light is absorbed by a colored chemical dissolved in the solution. This allows us to calculate the concentration or purity of chemicals, analyze specific chemical characteristics, and follow and measure chemical reactions in real-time.
As spectrophotometers use colored light to measure the absorbance of colored chemicals, let’s briefly describe the basic principles of color and light absorbance.
The colors we see are light waves of electromagnetic radiation and each color has a different wavelength. The wavelengths of light we can see (visible) are between 380 nanometres (nm) in the violet range (V) to 750 nm in the red range (R). Figure 1 shows the visible spectrum.
Figure 1: Visible light spectrum. Numbers represent wavelength (nm) (source: Wikimedia Commons, public domain).
Colored chemicals have color because they absorb some light waves while reflecting others. So, a red chemical absorbs all light waves and reflects the red wavelength light back to our eyes. And, a blue chemical reflects all light except for light with blue wavelengths.
Figure 2 shows the basic components of a spectrophotometer. Essentially, light from a specific source is split into different wavelengths (colors), then a selected wavelength passes through the sample and its final intensity is measured. Let’s break down each component.
Figure 2: Basic components of a spectrophotometer (source: Wikimedia Commons, CC-BY-SA).
The light source for a spectrophotometer that measures visible light uses an incandescent lamp with a tungsten filament. They look like very small light bulbs with two metal prongs.
The monochromator takes the light from the lamp and splits it into different colors or wavelengths. It works like a prism that bends light, separating the light into different wavelengths by refraction.
The aperture is simply a hole that creates a light beam. The size of the hole can be adjusted larger or smaller.
The cuvette is a small square vial that holds the solution with the colored chemical we want to measure (sample). They can be made of glass or plastic. They can hold volumes from 0.1 ml to 5ml.
The photodetector is a device that can convert light into an electrical signal. And the amplifier boosts the electrical signal to increase the sensitivity.
Spectrophotometers have a numerical readout to quantify the amount of light that’s absorbed as it passes through the sample. Older models use an analog needle and dial while newer models have digital readouts.
Spectrophotometers are used to measure the amount of light that’s absorbed by a colored chemical in a solution. But how do we convert the numbers on the output into usable information? To answer this question, we must understand the optical theory of absorbance described by the Beer-Lambert Law.
The Beer-Lambert Law describes the relationship between absorbance and the amount of chemical in solution and the distance light has to travel through the sample.
The Beer-Lambert Law is A = εcl, where A is the absorbance (no units), ε = molar extinction coefficient (M-1 cm-1), c = concentration of the substance (M), and l = path length of the light (cm).
Spectrophotometers have a panel area to set wavelengths, adjust other functions, and the output display. And they have a sample area with a square sample holder that nicely fits a cuvette, sitting directly in the path of the light beam shining on the detector. The sample area is also closed off to the outside light to prevent extra light from affecting the readings.
If we want to know the absorbance of a specific chemical in a solution, we must also account for the absorbance of the solution itself. So, we make two cuvettes, one with solution only (reference or blank), and one with solution plus the chemical we want to measure.
The reference solution is first put into the sample holder. The spectrophotometer has a button to “tare” the absorbance reading to A=0. Then the sample is put into the holder and the absorbance of the chemical is read on the display.
The absorbance spectrum of a chemical is the amount of light it absorbs at all the different wavelengths from violet to red. The spectrophotometer measures the absorbance of each different wavelength by adjusting the monochromator. Depending on the color of the solution, the absorbance will peak at specific wavelengths.
For example, figure 3 shows the absorbance spectrum of two types of chlorophyll (a,b), the biochemicals that make plants green. The plot shows the absorbance vs. wavelength(nm). We see absorbance peaks at either end for the light spectra. Chlorophyll absorbs blue and red light and reflects green light.
Figure 3. Absorbance spectrum of chlorophyll-a,-b (source: Wikimedia Commons, CC-BY-SA).
Measuring the concentration of a chemical in a solution using a spectrophotometer is its most common application. By rearranging the Beer-Lambert equation (A = εcl) to c= A/εl, we can calculate the concentration directly from an absorbance reading. Since the path length (l) is 1 cm, all we need is the extinction coefficient (ε). Extinction coefficients are typically already known so it’s easy to just plug the number into the equation and calculate concentration.
For example, the ε for chlorophyll-a is 105 M−1 cm−1. Therefore, a reading of A=1.0 would mean the concentration of chlorophyll = 1.0/105 = 0.00001 Molar (M) or 0.01 mM (or 10µM).
An extinction coefficient can also be determined from an absorbance spectrum. In figure 3, there are two absorbance peaks for chlorophyll-a, one in the blue region (430nm) and the other in the red region (662nm). If a known concentration of chlorophyll-a is measured, then ε (at 430nm or 662nm) can be calculated by ε = A/c.
Chemical kinetics is the study of reactions over time. Using a spectrophotometer, we can monitor a chemical reaction as it occurs in real-time. In the same way, we add a sample with one reactant to the cuvette and set the reading to zero. Then we add a second reactant, mix, and take absorbance readings of the product at various time points. By converting absorbance to concentration, we can plot the concentration versus time and calculate the rate of the reaction. And, by changing the concentrations of reactants, we analyze the nature of the chemical reaction itself.
We’ve discussed spectrophotometers that use visible light to measure colored chemicals. There are also spectrophotometers that use ultraviolet light (UV), and some that use more than one light beam.
Most visible spectrophotometers also use UV light to measure non-colored chemicals that absorb UV light using wavelengths between 180nm to 400nm. Instead of a tungsten lamp as the light source, UV spectrophotometers use a deuterium lamp (D2). Also, special cuvettes made from quartz are needed as glass and most plastic cuvettes absorb UV light. There are, however, UV cuvettes made from special plastics.
The most common use for UV spectrophotometer is to measure the concentration of DNA and proteins in solution. DNA absorbs UV light at 260nm while proteins absorb UV light at 280nm.
Single beam spectrophotometers have a single beam that shines through the sample. Before the sample is read, however, a reference sample must be measured. With a double-beam spectrophotometer, there are two beams, one for the reference sample and one for the test sample measured at the same time.
The primary limitation of spectrophotometers is they can’t measure chemicals that don’t have a color or absorb UV light. We can, however, add chemicals that react with uncolored compounds to produce a colored product that can be measured.
Spectrophotometers work best with dilute solutions that have an absorbance reading between 0 and 1. As described above, a sample that transmits only 10% of the light (90% absorbed) gives a reading of 1. Likewise, a more concentrated sample that absorbs more light and transmits 1% of the light (99% absorbed) gives a reading of 2. So, the most precise readings of absorbance are between 0 and 1 (0 to 90% absorbance). If a sample is too concentrated, it must be diluted, the absorbance read, then multiplied by the dilution factor.
Fortunately, spectrophotometers are built to need very little maintenance. Apart from keeping them clean and wiping up from liquid spills, the light sources need to be changed when they no longer produce enough light. It’s best to always have extra lamps on hand in case the lamps burn out.
Spectrophotometers are one of the most useful scientific instruments to detect, measure, and characterize chemicals in solutions. With chemicals that absorb light, their absorbance spectra and concentrations can be determined quickly and easily. And they can also be used to measure chemical reaction rates in real-time.
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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