Zone Preference in the Visual Water Maze
June 26, 2025
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The estimation of proteins via the quantitative method is one of the basic requirements in biochemistry. Proteins, from various perspectives, are substantially more complex than nucleic acids. Thus, it has been hard to give laboratory protocols that can be applied to proteins. The precise quantitation of the amount of protein during the steps of protein preparation is the only valid method to check the overall value of a procedure.
LOWRY protocol has been the most widely used method to estimate the number of proteins in biological samples. Lowry et al. (1940) was the biochemist who developed the reagent. Copper ion in alkali solution pre-treats the proteins followed by the reduction of phosphomolybdate phosphotungstic acid present in the Folin reagent. The end product of this reaction has a blue color.
Note:
This protocol is slightly modified, with respect to the original paper by Lowry, to work with smaller volumes.
Step 1:
Start the dilution of samples to an estimated 0.025-0.25 mg/ml with buffer. It is better to prepare a range of 2-3 dilutions spanning an order of magnitude if the concentration can’t be estimated. Prepare 400 microliters each dilution.
Step 2:
Prepare a reference of 400 microliters buffer. Prepare standards by adding 40-400 microliters to 13 x 100 mm tubes from 0.25 mg/ml bovine serum albumin + buffer to bring volume to 400 microliters/tube.
Step 3:
After adding 400 microliters of 2x Lowry concentrate, mix it thoroughly, and incubate at room temp. 10 min.
Step 4:
Add 0.2 N 200 microliters Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be achieved rapidly to avoid decomposition of the reagent before it reacts with protein. Incubate for 30 min. more at room temperature.
Step 5:
Use polystyrene or glass cuvettes to read the absorbances at 750 nm and after centrifugation, use an aliquot for protein determination.
Recording of absorbances should be done within 10 minutes of each other for this modified procedure. This modification is more sensitive to protein than the original and less sensitive to interfering agents. Proteins with an abnormally low or high percentage of tryptophan, tyrosine, or cysteine residues will give high or low errors, respectively.
This modification method by Sargent (1985) is also used in the quantification of proteins. The principle of this method includes a 50-fold increase in sensitivity with respect to the Lowry standard protocol. It is possible to estimate 0.1– 1 µg protein and 4–40 µg/ml, respectively.
Step 1:
Start the dilution of samples to an estimated 0.025-0.25 mg/ml with buffer. It is better to prepare a range of 2-3 dilutions spanning an order of magnitude if the concentration can’t be estimated. Prepare 400 microliters for each dilution.
Step 2:
Prepare a reference of 400 microliters buffer and add 400 microliters of 2x Lowry concentrate, mix thoroughly, and incubate at room temp. 10 min.
Step 3:
Add 0.2 N 200 microliters Folin reagent very quickly, and vortex immediately. Complete mixing of the reagent must be achieved rapidly to avoid decomposition of the reagent before it reacts with protein. Incubate for 30 min. more at room temperature.
Step 4:
Extract the samples twice with 1 ml ethyl ether each. After centrifugation remove the ether by aspiration; remove the remaining ether in the aqueous phase with a SpeedVac.
Step 5:
Now add Soln. E, i.e. Malachite green and note the observations.
Measure the absorbances at 690 nm immediately after the addition of solution E and prepare the standard curve in the range between 0 and 1 µg BSA.
The microassay method is used for samples with low protein concentrations. The 96 well plate assay is used to perform the Lowry assay in a plate format.
Start the dilution of the sample with NaOH to a final concentration of about 0.1 moles NaOH and to an amount of protein within the measuring range.
Step 1:
Pipette 40 µl of each unknown (25 µl) and standard sample replicate into a microplate well. The same buffer that was used in the samples will be used for the diluents.
Step 2:
After adding 200 µl of freshly prepared Lowry Assay Mix, immediately mix the microplate for 30 seconds.
Step 3:
For exactly 10 minutes, incubate the microplate at room temperature.
Step 4:
After adding 20 µl of freshly prepared Lowry Working Solution to each well, immediately mix the microplate for 30 seconds.
Step 5:
Incubate 30 min at room temperature. Measure the absorbance at 620nm-660 nm on a plate reader.
If all the samples are read at the same time, the absorbance will not change significantly. The Lowry assay is not an endpoint assay, so samples will change in absorbance if too much time elapses between sample readings. During the reading of samples, i.e. less than 10 minutes, the typical time that elapses does not ordinarily result in significant changes in absorbance.
The Lowry protein assay is a susceptible but profoundly nonspecific procedure. The standard Lowry protein assay has been adjusted so that protein can be measured in the presence of interfering chemicals. The method depends on the observation that in the presence of 125 ìg/ml of Na-deoxycholate, bovine serum albumin (5—50 ìg) is reproducibly precipitated (90–104%) by 6% trichloroacetic acid. Interference by glycerol, sucrose, EDTA, Tris—HCl, and Tricine can be eliminated. If the NaCl concentration is adjusted to 1 m, the protein samples containing carrier ampholytes can also be assayed.
Step 1:
Dilute 5 to 100 µg of protein and standard, respectively, with ddH2O to 1.0 ml.
Step 2:
After that, add 0.1 ml of Soln. A.
Step 3:
After a further 10 min at RT add 0.1 ml of Soln. B. After mixing the solution well, place the samples for centrifugation with 3000 × g at RT 15 min later.
Step 4:
Now resolve the precipitate in 1.0 ml ddH2O, and then add 1.0 ml Soln. G.
Step 5:
Now the precipitate should be resolved entirely. After further 10 minutes add 0.5 ml of Soln. H, mix well and read the absorbances at 700 nm 30–45 minutes later.
In the presence of 125 μg/ml of Na-deoxycholate at 700 nm, bovine serum albumin (5—50 μg) is reproducibly precipitated (90–104%) by 6% trichloroacetic acid.
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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