Factors that Influence Experimental Outcomes and How to Overcome Them

Factors that Influence Experimental Outcomes and How to Overcome Them

Experimental outcomes can be influenced by a variety of factors, some of which can be controlled for. Minimizing confounding factors is crucial to gathering reliable and repeatable results.

One of the biggest issues in animal research today is the replicability of results. Too often animal study outcomes can not be repeated. This is not hard to believe given that the use of animals themselves provides inherent variability, even when all other factors are controlled for. Differences in the strain of animals used, as well as the age of the animals, time of day that experimental tests are administered, and how long the animals were handled prior to experimental testing are just some of the factors that can impact experimental outcomes.

While it is impossible to eliminate all external factors, animal-experimenter interactions can have a huge impact on results and should, therefore, try to be minimized as much as possible.

How can you mitigate animal-experimenter interactions?

At AMUZA, we offer a variety of automated behavioral tests that were designed specifically to improve the reliability and repeatability of behavioral assays.

For example, our Touch Panel operant training system is an automated operant chamber that utilizes photo beam sensors in the touch panel itself to improve the accuracy of responses from small rodents. The Touch Panel also includes software that enables users to design and run their own tasks with video tracking capabilities for automated data collection.

Even our standard mazes come with video tracking and automated data collection and analysis.

Touch Panel

Self Head-Restraining Platform

Furthermore, one of our other products, the Self Head-Restraining Platform, was designed to completely automate the head-fixation process in mice in order to streamline head-fixed behavioral assays.

In fact, the platform, originally developed by Dr. Andrea Benucci at RIKEN brain institute, was designed specifically to help overcome the reproducibility crisis.

Not only do our tools free up experimenter time and labor to focus on the actual science, they help remove unwanted experimenter bias by standardizing the experimental testing arena.

Even with automated behavioral tasks, however, it is still possible to introduce experimenter bias. This is why we also recommend that you perform rodent behavioral tests at roughly the same time each day, as well as handle experimental animals equally. Ideally, the same experimenter should be handling the animals each day. If this is not realistic, different experimenters should be counterbalanced across days, or across testing groups.

Also, if you plan to use different strains of mice or rats for your experiments, make sure to run behavioral tests across these different strains to account for any strain-specific differences.

Additionally, with our automated rodent behavior systems, we recommend that the motivation of the experimental animals to perform the task is consistent. If animals are food or water-deprived, weights should be taken daily initially and then weekly thereafter to ensure that test subjects are maintained at similar percentages of their free-feeding body weight.

Cleaning the testing chambers between use

All of our behavioral tests are made out of acrylic that is easy to clean as well as removable floors. Testing arenas should always be cleaned between experimental sessions to make sure the scent of the previous animal will not influence behavioral results.

For more detailed information about our automated behavioral tests connect with us today.

Mice Learn Voluntary Head-Restriction

Mice Learn Voluntary Head-Restriction

In this video, we will walk you through how exactly mice learn to self head-restrain using the self head-restraining platform. The concept is quite simple and relies on operant or classical conditioning principles. Mice learn head-restriction by walking through the narrow corridor of the chamber past a set of rails that latch the head plate in place allowing the mouse to receive a water reward.

The rails are locked into place once the photo beam sensors, located on the corridor, are triggered when the mouse has reached the correct position. The rails can be set to lock for a specified amount of time, while the mouse performs a behavioral task.

In this case, the timer has been set to 10 min. After 10 min the rails will automatically release, allowing the mouse to return to its home cage.

The most critical step in learning self-latching is the habituation phase.
During the habituation process, which typically takes between 1-2 weeks, a habituation tube with a similar non-locking rail system is placed in the home cage and attached to a water reward. At this time, mice should be placed on water restriction so that they are motivated to seek water. Mice gradually approach and enter the tube, following the rail system until they are able to obtain water.

The key difference between habituation and the actual task is that during habituation the rails do not automatically lock so the mouse can exit at any point. This allows mice to get comfortable with having their head plates in the rail system but also gives them the opportunity to voluntarily escape.

After habituation, once mice are comfortable entering the corridor and sliding their head plate into the rails they easily learn to self-latch using the self head-restraining platform and can then be trained to perform operant behaviors while head-restrained.


A tool to Combat the Current Reproducibility Crisis in Neuroscience

A tool to Combat the Current Reproducibility Crisis in Neuroscience

The Problem:

Reproducibility, stemming from the inability to replicate scientific studies across labs, is a major issue plaguing Neuroscience research today and is particularly prevalent in animal studies. This is not hard to believe given that the use of animals themselves provides inherent variability, even when all other factors are controlled for. Differences in animal handling, even by the same experimenter can also negatively impact experimental outcomes. 

In addition, advancements in technology have raised the bar for animal experiments. It is now possible to examine neural activity in real-time as animals perform behavioral training during head-restriction.  However, these techniques require extensive training of animals by the experimenter which greatly limits throughput, making it challenging and time-consuming to conduct high powered studies with sufficient sample sizes. Underpowered studies, even those that are technologically advanced, are futile for producing results. 

The Solution:

Our behavioral system, the Self Head-Restraining Platform – originally developed by O’Hara and Dr. Andrea Benucci at RIKEN Brain Institute, was designed specifically to overcome both the issue of experimenter bias and small sample size by enabling automatic mouse head-restriction. 

How it Works:

The Self Head-Restraining Platform essentially teaches mice to train themselves to perform head-restricted operant behavior. It is completely automated, attaches to the home cage, and can run around the clock without experimenter supervision. This not only removes experimenter bias, it greatly increases data throughput, making it manageable to run studies with sufficient sample sizes.

Dr. Andrea Benucci is currently using 12 setups to effectively train 48 mice to perform head-restriction at a time!

“Previously, training just one mouse took about 15 hours of researchers time. Now with 12 setups, we are down to less than one-and-a-half hours.” – Andrea Benucci.

Learn more about the Self Head-Restraining Platform.

How it Works: Steps to Voluntary Head-Restriction
with the Self Head-Restraining Platform 

The sequence of steps leading to head-restriction. The head plate (black bar on the mouse’s head) is progressively restrained into rails that get narrower as the mouse moves forward(1,2). The forward motion of the head plate mechanically lifts the latching pins, which are then lowered back down due to gravity (3,4). Continued forward movement by the mouse then lifts and lowers the second set of pins (4). After the session ends, a computer-controlled motor lifts up both pins and releases the mouse ( adapted from Ohara & Co., LTD & Aoki et al., 2017).

To see the Self Head-Restraining platform in action
contact us for video access.

Part II – Next stop – RIKEN Center for Brain Science in Tokyo, Japan.

Part II – Next stop – RIKEN Center for Brain Science in Tokyo, Japan.

Next, I had the pleasure of visiting Dr. Andrea Benucci’s lab at RIKEN Center for Brain Science Institute. Dr. Benucci and O’Hara teamed up to design the Self Head-Restraining Platform, a high throughput system for mouse behavior and neurophysiology.

Teaching mice to perform head-restrained behavioral tasks during neural recording has proven extremely laborious and time consuming, making such experiments extremely low throughput.

According to Dr. Benucci, “As a post-doc, I spent months training individual mice to perform head-restrained behavioral tasks during two-photon imaging sessions, and thought there has to be a better way!”

Dr. Benucci felt compelled to find a solution to this problem. His answer was the Self Head-Restraining Platform. With this system, mice learn to voluntarily head-restrain themselves into a self-latching device in order to receive a water reward. The platform affixes to the home cage, and the dual-cage platform can train up to 4 mice per day.

Me with Dr. Andrea Benucci in his testing room, where he currently has 12 working platforms!

Dr. Benucci has 12 platforms set up in his lab to effectively train 48 mice per day! Once mice are trained to head-restrain (1-2 weeks), they are then trained on a complex visual discrimination task and imaged under a two-photon microscope. With the self-latching system, mice can be imaged for up to 20 minutes at a time.

To learn more about Dr. Benucci’s research check out his website.

You can find the full paper on the Self Head-Restraining Platform published in Nature on the Self Head-Restraining Platform product page on our website!

Stay tuned for my next stop – Kyoto University!

Teaching Mice to Train Themselves : The Scoop on O’Hara’s New Behavioral Testing System

Teaching Mice to Train Themselves : The Scoop on O’Hara’s New Behavioral Testing System

Understanding how neural circuits give rise to behavior is one of the most sought after questions in Neuroscience. With O’Hara’s new behavioral testing system, finding an answer just got easier

O’Hara teamed up with researcher Dr. Andrea Benucci at the RIKEN Brain Science Institute in Japan to develop a high-throughput and fully automated system, The Self Head-Restraining Platform, to simultaneously assess mouse behavior and neurophysiology.

Mice learn to voluntarily self-restrain their heads for a reward, enabling neural recording simultaneously during behavior.

Training head-restrained mice to perform complex tasks is extremely labor and time-intensive, leaving little time for actual research.

“Previously, training just one mouse took about 15 hours of a researcher’s time. Now, with twelve setups we are down to less than one-and-a-half hours.” – Andrea Benucci.

Using the Self Head-Restraining Platform, mice can engage in operant tasks at-will, without any intervention from the experimenter. The system has already been used to effectively train 100 mice.

In addition to the platform being high-throughput, mice learn to self-stabilize their heads,enabling an easy transition to in vivo examination of neural dynamics during behavior.

“Normally we see a decline in mouse performance or other incompatibilities when moving from highly-trained behaviors to different types of experiments for brain recordings, but that doesn’t happen with our system,” says Benucci.

Learn more about the Self Head-Restraining Platform by visiting https://amuzainc.com/operant-learning-self-restraining-platform/