Zone Preference in the Visual Water Maze
June 26, 2025
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The modern pipette has had a colorful history as a standard tool in the scientist’s arsenal. What began as the usage of straw, one’s mouth, and the scientific principles of suction is now one of the most technologically-evolved yet extremely straightforward devices in the modern laboratory.
Commonly used in genetic research, chemistry, microbiology, and pharmacological testing; pipettes and micropipettes are glass or plastic tubes used to measure, transfer, and deliver substances of accurate volumes. Most pipettes function by creating a vacuum above the space that the liquid shall fill and then control the uptake of the liquid by releasing this vacuum, suctioning the liquid upwards. Simple enough as this may sound, pipettes have come a long way–let’s look at its evolution through an exhaustive list of products that are widely used today.
Aside from his more popular invention of the process of pasteurization, we also have Louis Pasteur to thank for the first pipette ever invented. As the founder of medical microbiology and the proponent of Germ Theory, it was then important for Pasteur and his experiments that things be kept clean and germ-free: hence, the invention of the first pipette. The Pasteur pipette, which are the familiar eye droppers and chemical droppers that we still encounter in everyday use, was then merely designed to prevent contamination in transferring small amounts of liquids. The addition of rubber teats at the end of long, thin, glass tubes made aspirating and dispensing of liquids a quick and easy affair. Today, these pipettes are used for the transfer of rough, uncalibrated volumes of liquids of up to 2.5 milliliters.
Pinch the rubber teat of the Pasteur pipette with thumb and forefinger, and immerse the tip of the pipette on the surface of the liquid. Then, slowly release pressure on the rubber teat and wait for the liquid to go up. Once pressure is released, and the proper amount of liquid is aspirated, bring the full Pasteur pipette to the receiving container and pinch the rubber teat again to dispense the liquid on the side of the container’s wall.
Pasteur’s glass pipettes were a 19th-century hit, but problems arose from the fragility of its material. The invention of plastics in the 1940s, therefore, paved the way for a modification to the original pipette design–plastic Pasteur pipettes, also called transfer pipettes, were single pieces of molded plastic manufactured using the blow-molding process on low-density polyethylene (LDPE). Both the stem and bulb are part of the single piece of plastic, in place of the rubber attachment in glass pipettes. Today, these disposable transfer pipettes are made in a variety of sizes and shapes, and some include graduation marks for approximate volume calibrations. Note, however, that though transfer pipettes may be perfect for use with aqueous solutions, organic solvents such as acetone may dissolve the plastic material.
Transfer pipettes use a similar procedure as with the usage of the Pasteur pipette, but instead of a rubber teat, the plastic bulb is pinched.
The high-end and complex pipette varieties that offer more accuracy and precision than Pasteur and transfer pipettes emphasize on the other aspect of pipetting, aside from the transfer and delivery of liquids–measurement. And, though considered a reliable high-precision instrument, the pipette does not achieve accuracy and precision on its own. A tool is only as good as its user, and, in the laboratory context, the wielder of the pipette must be well-prepared and knowledgeable with common pipetting techniques to make full use of its features. More importantly, scientists are expected to make informed decisions when selecting which pipette to use. Which pipette is the right pipette? How are they used? Let’s look at our list again.
Volumetric pipettes resemble thin rolling pins with large bellies, blunt on one end and tapered on the tip. Typically, these pipettes are used to deliver single, specific volumes between 1 and 100 milliliters, at which they are calibrated. These glass pipettes offer more accuracy than Pasteur and transfer pipettes, emphasizing on the other aspect of pipetting, aside from the transfer and delivery of liquids–measurement. By having the middle bulge where the bulk of the liquid is stored, and the single volume marker at the thin portion, errors in accuracy are extremely lessened. The narrow diameter of the thin portion makes errors more obvious and easier to point out.
All micropipettes operate like an automatic syringe, but the two primary types–the air-displacement pipette and the positive-displacement pipette–differ based on the presence of an air cushion between the pipette piston and the liquid sample.
Air-displacement pipettes always have a cushion of air between the pipette piston and the liquid. The simpler of the two instrument types, air-displacement pipettes are easily influenced by factors such as temperature, atmospheric pressure, specific gravity, and the liquid’s viscosity. Keeping the liquid away from the piston or barrel is an advantage, but also provides many limitations in terms of maintaining accuracy. For instance, working with liquids that may easily evaporate would require the use of the other type of pipette–positive-displacement pipettes.
Air-displacement and positive-displacement micropipettes involve a quicker and more efficient procedure for transferring liquids of specific volumes. The first step to using these automatic micropipettes is the push-button volume adjustment. Hold the body of the micropipette in one hand and use the other hand to rotate the push-button. Do not attempt to force the volume setting beyond the limits of your micropipette, at the risk of internal damage. Also, make sure that the numbers of your volume setting are in proper alignment. Afterwards, a disposable pipette tip is attached.
After setting the volume and attaching the disposable tip, the next step is the process of pre-rinsing. Pre-rinsing, also called pre-wetting, is a highly-advised procedure for achieving greater uniformity and precision, as it provides identical contact surfaces for the liquid to be aspirated. To pre-rinse, simply aspirate with the tip and then dispense back into the original reservoir or to a waste receptacle. Pre-rinsing should be performed each time you change a pipette tip, and when you increase the volume setting.
The operation of automatic micropipettes involves two points of resistance — the first stop, the point at which the piston reaches the calibrated volume on the digital volumeter, and the second stop, at which the remaining liquid is fully expelled or purged. When aspirating, two factors can influence the accuracy of your measurement — the aspiration angle and immersion depth. Prepare the micropipette by holding it in a nearly vertical position and depressing the plunger or push-button to the first stop position using your thumb. Once the push-button is in the first stop, immerse the pipette tip just below the surface of the liquid, to avoid droplets sticking on the outside of the pipette tip and slowly and smoothly release the plunger or push-button back to the rest position as you wait for the liquid to move up and into the tip. Do not let the plunger snap back up as this will result in air bubbles.
Once the liquid is collected, place the pipette tip at an angle of 10 to 45 degrees against the inside wall of the receiving vessel, and depress the plunger smoothly back to the first stop position to dispense the liquid. Wait one second, and then depress the plunger once more into the second stop position to “purge” or “blow-out” the tip of any residual liquid. Finally, remove the pipette tip by sliding it off the sidewall. The procedure is the same for positive-displacement micropipettes, except both the disposable pipette tip and piston are ejected and replaced after each transfer.
The mechanism of positive-displacement pipettes involves a direct contact of the piston with the sample. This set-up makes positive-displacement pipettes more accurate for pipetting volatile solutions because there is no dead space for the liquid to evaporate. The absence of the air cushion also lessens the risk of contamination with corrosives and other bio-hazardous materials. However, these pipettes are more expensive as they replace both piston and tip after each use.
A crucial step in using automatic micropipettes is choosing the right pipette tip. Pipette tips, which hold the aspirated liquid provide efficiency in laboratory procedures by not requiring thorough washing or cleaning, through quick replacement of the reusable or disposable tips after every use. Multichannel pipettes, meanwhile, provide maximum efficiency by allowing the aspiration of multiple pipette tips at the same time.
Finally, like any working device, micropipettes have many moving parts that are susceptible to wear and tear or breakage. Therefore, any responsible owner of pipettes and micropipettes must exhibit regular care and maintenance, even when these tools are not in use. Preventive pipette maintenance includes the process of calibration in preserving high-quality performance, accuracy, and precision. It is important to have your pipettes serviced at least twice a year by experienced pipette handlers and pipette clinics. Pipette owners must also clean pipettes after every session, and check pipettes daily for damage before use. In storing pipettes, pipette holders are recommended to ensure that the instruments are kept vertical and upright.
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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