Conducting behavioral experiments requires careful consideration of various factors to ensure reliable and reproducible results. In addition to experimental manipulations like genetic alterations and drug administration, researchers must address five critical factors influencing animal behavior studies. Understanding and managing these factors can significantly enhance the validity and reproducibility of your animal model studies. Here, we’ll explore each of these factors and provide insights on how to effectively manage them.

Manage Environment for Enhanced Habituation in Behavior Studies

The success of behavioral experiments is heavily influenced by environmental conditions. Adhering to international standards for cage size, room temperature, humidity, food, water, and bedding replacement is crucial. For example, variations in lighting environments within cage racks can impact experimental results. Researchers should carefully record and control the location of animals within the rack, especially in relation to lighting. Environmental noise, such as ventilation systems, also plays a role, requiring researchers to understand and manage differences between living and experimental areas. Ideally, however not always practical, the experimental environment should closely replicate the home cage environment.

Handle Animals Appropriately to Minimize Stress

Regular handling of laboratory animals is common practice, but behavioral investigators must take extra precaution. Habituating the animals to the investigator by handling animals daily, starting at least a week before the experiment, can mitigate unnecessary anxiety and fear and ensure more accurate data. During transitions from cages to experimental settings, gentle and prompt handling is vital. Avoiding chasing, grabbing, and minimizing midair hanging by tails is essential for maintaining the well-being and reliable behavior of the animals.

Pre-Experiment Episodes and Waiting Periods

Introducing a holding area and a specified waiting time before the experiment can reduce data variability. This strategy ensures uniformity in the episodes leading up to the experiment for all animals. The holding area, secluded from the investigator’s view, should offer a quiet environment with consistent lighting to avoid disrupting the animals. Transferring the animals from the home cage environment to the experimental environment can be stressful. Allowing for at least 30 minutes in the holding area will allow the animals to calm down following room transfer. This structured approach contributes to more consistent and reliable behavioral outcomes.

Timely Execution of Experiments

Consistency in the timing of experiments is crucial, especially for studies conducted over multiple days or involving repeated training sessions. This is particularly important as many investigators run behavioral assays during the animal’s dark (night) cycle, maintaining the same schedule each day will minimize disruptions to the animal’s sleep and increase the consistency of their performance on a given task. Following a rigid experimental schedule helps maintain stability and reduces potential confounding variables, contributing to the accuracy and reproducibility of results.

Maintaining Consistent Experimental Conditions

Acute attention to experimental conditions is also imperative. Aim to keep variables such as temperature, humidity, sound, and light consistent. Many research labs can be noisy environments, so it is important to consider the use of soundproofing solutions, such as soundproof boxes or rooms, to minimize external influences. However, be mindful of creating environments that are too silent, as this can induce unnecessary anxiety. Providing background sounds resembling the animals’ normal living environment can strike the right balance between control and ecological validity.

By addressing these five key factors, researchers can optimize the well-being of their animals and conditions of their behavioral experiments, leading to more reliable, valid, and reproducible outcomes in animal model studies.

If you would like to download these factors in a convenient application note format or are interested in learning more about additional behavioral neuroscience applications and other techniques such as Fiber Photometry and Optogenetics, please take a look at our application note and eBook page on our website.

If you are interested in studying the emotional or motor activities of animals, such as anxiety, exploration, or locomotion, you might want to consider using an open field test. This is a simple and widely used method that involves placing an animal in a large, empty box and observing its behavior. In this blog post, we will give you some tips on how to choose the right dimensions, materials, and light conditions for your open field test.

Dimensions

The size of the experimental box should be large enough to allow the animal to move freely and explore the environment, but not so large that it becomes stressful or overwhelming. The box should also be higher than the home cage to prevent the animal from escaping.

For mice, a suitable box size is at least 400 x 400 x 300 (h) mm, but you can also use a larger box, such as a 500 x 500 x 300 (h) mm box, which is ideal for open-field tests in mice. For rats, the standard box size is 900 x 900 x 500 (h) mm, but you can also use a smaller box, as long as it is larger than 600 x 600 x 500 (h) mm.

Materials

The inner walls and floor of the box should have a matte finish to reduce light reflection and glare, which can affect the animal’s vision and behavior. The box should also have a non-slippery surface to provide adequate grip for the animal and prevent falling or sliding.

While some animals may prefer wood floors and show more activity on them, wood is not a good material for open field tests because it can absorb odors and germs and cannot be easily cleaned or disinfected. Therefore, we recommend using plastic or metal boxes that can be sanitized after each use.

Another factor to consider is the transparency of the box walls. Some researchers may prefer transparent walls to observe the animal better, but this can also expose the animal to external visual stimuli, such as the experimenter, other animals, or objects, which can influence its behavior. Therefore, we recommend using opaque walls or covering the transparent walls with curtains or screens to create a more isolated and controlled environment. We also suggest using matte light gray walls and floors, as this color can help the animal acclimate faster to the new setting.

Light Conditions

The amount and quality of light in the box can also affect the animal’s behavior. Generally, animals tend to be more active and exploratory in dimmer light conditions, as they feel less exposed and threatened. In contrast, brighter light conditions can induce more anxiety and inhibition in the animal, as they feel more vulnerable and exposed.

In recent years, some researchers have reduced the light intensity in their open field tests to less than 10 lux, which is equivalent to a dark room with a faint light source. They have found that this low light level can increase the animal’s movement and exploration, compared to the traditional 50 to 200 lux, which is equivalent to a well-lit room or a cloudy day.

However, it is important to ensure that the light is evenly distributed in the box, as any contrast between light and dark areas can create a preference for the dark areas, where the animal feels more secure and hidden. To achieve uniform illumination, we recommend using LED light panels with dimming functions that can be placed on top of the box. This method can also allow you to adjust the light intensity according to your experimental needs.

Sound Environment

One of the challenges of conducting an open field test is to control the background noise in the room where you perform the experiment. Noise can interfere with the animal’s behavior and affect their responses. For example, sudden or loud noises can startle them and make them more anxious or less active.

To avoid this problem, you need to create a consistent and neutral sound environment that masks other noises in the room. One option is to use a white noise generator that produces a constant and uniform sound of 60 decibels. This can help to reduce the variability and unpredictability of the noise level and create a more stable and calm atmosphere for your animal subjects.

However, if you cannot provide the same sound environment every day, or if there are other sources of noise that you cannot eliminate, you may need to use a soundproof box or place the experimental box in a soundproof room. This can help to isolate your animal subjects from any external noise and ensure a more controlled and quiet environment for your experiment.

Experiment Duration

Another factor that you need to consider is how long you should run your open field test. The duration of the test can affect the quality and quantity of your data, as well as the welfare and comfort of your animal subjects.

Some older papers suggest that an open field test should last for 5 minutes, but this may not be enough to capture the full range of your animal’s behavior and activity. More recent studies have shown that movement often increases after the first 5 minutes, as your animal subjects become more familiar and comfortable with the environment. Therefore, it is recommended that you extend your open field test to at least 10 minutes, to allow for more exploration and adaptation.

However, if you want to measure more long-term effects or changes in your animal subjects’ behavior and activity, you may need to run your open field test for longer periods, such as 2 hours. This can help you to observe how your animal subjects respond to prolonged or repeated exposure to the same environment, and how they develop habits or preferences over time.

But remember, the longer you run your open field test, the more you need to take care of the animal’s well-being and comfort. You need to provide them with adequate food, water, and bedding, and monitor them for any signs of stress, fatigue, or injury. You also need to ensure that the temperature and humidity of the room are appropriate and consistent.

Experimental Protocol

To start the test, you need to place the animal in a specific spot in the box and let it roam freely for a certain amount of time. It is important to be consistent and use the same spot and posture for all animals, so you can compare their results fairly. A good spot to use is near the wall, because animals that are nervous or anxious tend to stay close to the edges, while those that are more confident or adventurous venture into the center.

As the animal explores the box, you can observe and record various aspects of its behavior, such as how much it moves, how often it turns, how long it stays still (freezing), and how many times it stands up on its hind legs(rearing). These indicators can tell you a lot about the animal’s emotional state, curiosity, and motivation. For example, animals that move a lot, turn frequently, and rear often are usually more active, alert, and interested in their surroundings, while animals that move little, turn rarely, and rear less often are usually more passive, calm, and cautious which may indicate an anxiety-like state.

To increase the accuracy and convenience of data analysis, you can use a more advanced Open Field system, which automatically measures and analyzes the animal’s behavior using image processing and/or infrared beams. This approach also allows for automatic detection of rearing. However, keep in mind that different researchers may have different interpretations of what these behaviors mean, so you should always focus on the changes in the frequency and duration of these behaviors, rather than their absolute values.

After the test is over, you need to remove the animal from the box as quickly as possible, to avoid stressing it or affecting its behavior. You also need to clean and disinfect the box before testing the next animal. This is to prevent any contamination or odor that could influence the animal’s behavior. If you are using mice, especially the C57/B6 strain, you should avoid using alcohol to disinfect the box, because they are very sensitive to it. You should wait until the alcohol evaporates completely, or use a different disinfectant, before testing the next mouse.

Summary

While the Open Field test method remains an invaluable tool for researchers studying a wide range of behaviors. It is important to take the proper measures to reduce environmental and experimenter bias in your results. By following the above guidelines, you can conduct open field tests with precision and reproducibility, ensuring the collection of reliable data on animal motor function and emotional states. If you are interested in learning more about additional behavioral neuroscience applications and other techniques such as Fiber Photometry which can be paired with behavioral assays, please take a look at our application note and eBook page on our website.