Snapshot: What is Gait Analysis in Ataxia Mouse Models?

A key role of the cerebellum is to control and fine-tune coordinated movement such as walking. Although walking is an unconscious behaviour, it is actually very complex and requires many systems to work together. The specific mannerisms and patterns of coordinated movement that make up how an individual walks are called gait.

Since ataxia affects cells in the cerebellum, many ataxia patients exhibit a change in their gait. This change can reduce their mobility and be disruptive to daily life. Analyzing gait using behavioural experiments in ataxia mouse models helps researchers to better understand the disease. But how exactly does one study gait patterns in a mouse?

Black rat walking in front of white background
Photo used under license by Eric Isselee/Shutterstock.com.

Traditionally, a researcher measures gait by performing a footprint analysis that uses non-toxic water-coloured paint and a long strip of white paper. The front and back feet of the mouse are dipped into two different colours of paint. Then the mouse then runs across the paper leaving a trail of coloured footprints to be analyzed. This allows for several gait measurements to be taken. From this, the researcher can then determine how ataxia changed the mouse’s gait. For example, a researcher can look at if the mouse takes shorter strides than a healthy control or whether the mouse tends to walk in a more crooked manner.

Although easy to perform, footprint analysis is time-consuming and highly prone to error as the experimenter does all measurements with a ruler by hand after the mouse has run. Since gait is a complex action with many variables, some subtle differences may be difficult to detect this way. Luckily, researchers have developed several digital gait analysis systems, such as the DigiGait, CatWalk, and TreadScan systems. These digital gait devices make use of transparent corridors with cameras underneath that allow the researcher to record the running behaviour of the animal. Researchers then use software to automatically detect and analyze the footprints.

One lab has taken gait analysis even further. They developed a method to detect extremely subtle differences in gait that the human eye cannot detect. This technique, called LocoMouse, was developed by the Carey lab to analyze patterns of limb movements, rather than simply footprints. LocoMouse utilizes artificial intelligence to recognize and analyze the movement of limb, tail, and head position in a walking mouse.

Using this, the Carey lab has shown a significant difference between a healthy mouse and one with ataxia. Most importantly, the method also detects differences between different ataxia mouse models. By uncovering subtle differences in gait, researchers may better understand the different underlying physiological changes in the cerebellum in different ataxias.

It should be noted that a key pitfall of studying gait in mice is that they are four-legged while humans walk on two legs. This is important, and means that the variables that affect gait in a mouse will be different than those of a human. There may not be a direct correlation between gait changes in ataxia patients and gait changes in mice. That being said, gait analysis remains an important tool in the ataxia researchers’ toolbox. It will continue to provide critical insight into how ataxic physiology affects behaviour.

If you would like to learn more about gait analysis in mice, take a look at these resources by the Noldus Information Technology and Mouse Specifics Inc.

Snapshot written by Eviatar Fields and edited by Dr. Chandana Kondapalli.

Snapshot: What is the Morris Water Maze Test?

Spinocerebellar ataxias (SCAs) are well known for worsening motor coordination symptoms caused by the degeneration of the cerebellum. Yet, increasing reports indicate that broader changes are occurring in the brains of some SCA patients. This includes changes in the hippocampus, a brain region critical for learning and memory. One way to test learning and memory in mice is the Morris Water Maze Test. Researchers use this test on SCA mouse models to investigate how and when learning and memory symptoms arise. More importantly, we can also use this test to evaluate the effect of potential treatments on learning and memory.

white mouse swimming with its head poking up above the water
Although mice can swim quite well, they don’t like swimming. The Morris Water Maze takes advantage of this to test the learning and memory of mice. Photo used under license by Aleksandar Risteski/Shutterstock.com.

The Morris Water Maze consists of a large circular pool of opaque water. A platform is placed in the pool just under the surface of the water so that the mouse won’t be able to see it. Though mice are good swimmers, they don’t particularly enjoy swimming. Mice will always attempt to find the platform as quickly as possible. Shapes on the walls around the pool help the mice orient themselves within the pool (first panel in the figure below).

The first time a mouse swims in the pool (second panel in the figure), the mouse tends to swim aimlessly around until they eventually find the hidden platform. Each subsequent time the mouse swims in the pool, the mouse will get better and better. Using the shapes on the wall to help identify where they are in the pool, the mouse will eventually learn and memorize the platform’s location.

First day, mouse does not know wehere the plaform is an swims a lot. Second day, the mouse still swims a while but remembers where the platform is. On the last day, the mouse knows where the platform is and goes right there.
The three steps in the Morris Water Maze. Image made by Larissa Nitschke use BioRender.

As that happens, they will be better and better at the task. Eventually, the mice will swim immediately to the platform when placed in the pool (third panel in the figure). Researchers can measure this improvement by measuring how much time it takes the mouse to reach the platform and the length of its path to the platform. Additionally, to assess the strength of the memory, researchers can take out the platform from the pool in what is called a “probe trial”. Mice that spend more time in the area where the platform used to be are considered to have built the strongest memories of that location.

As is the case for some SCA mouse models, mice with impaired learning and memory have more difficulty learning and remembering the correct location of the platform. As a result, they spend a longer time searching for and swim longer distances to the platform. Overall, they display a poorer improvement over time. By using the Morris Water Maze Test on SCA models that receive different treatments, scientists can then further test which therapy could improve their learning and memory symptoms. Therefore, the Morris Water Maze Test may help identify new therapeutic strategies to treat learning and memory problems in patients.

If you would like to learn more about the Morris Water Maze, take a look at these resources by the Scholarpedia and JOVE.

Snapshot written by Carrie Sheeler and edited by Dr. Larissa Nitschke.

Snapshot: What is the balance beam test?

When you think of a balance beam, you might think of gymnastics. For humans, a balance beam is a surface where we perform jumps, flips, and other athletic feats. Whether it’s a child taking their first class, or an Olympic athlete going for gold, the balance beam requires both balance and coordination. When a scientist puts a mouse through the balance beam test, they don’t ask them to do this kind of complicated routine, but they are testing those same abilities.

Little Black Mouse on a White Background
Little Black Mouse on a White Background. Photo used under license by Michiel de Wit/Shutterstock.com.

The equipment setup for the balance beam test is simple: two platforms with a beam running between them plus lots of padding underneath so the mouse doesn’t get hurt if it falls off. Over multiple days, the scientist will train the mouse to run across the beam from one platform to another. Once the mouse has been trained, it will go through multiple official test runs. In these tests, the scientist will measure the time it takes for the mouse to cross the beam. They will also count the number of times one of its paws slips off the beam during the crossing. You can see some videos of mice doing the test here.

Mice that have problems with balance and coordination usually take longer to cross the balance beam and have more paw slips during the crossing. The mice might take longer to cross because they are clinging to the beam to try to stay on. Their paws might slip more because they cannot coordinate their movements properly. The scientist can also compare the measurements from the first day of training with the measures taken during the official runs. This shows how well the mouse learned to stay on the beam. This is useful because learning how to do a task and performing the task are two different things. Some parts of the brain are more important for learning, while others are more important for doing the task. Thus, telling those two aspects apart can be useful.

Mouse cossing a balance beam connecting two platforms

A typical balance beam setup, with two platforms and a beam between them. Image by Amy Smith-Dijak.

The balance beam test has been used to understand balance and coordination in both healthy mice and mouse models of disease. In healthy mice, scientists studying the basic biology of balance and coordination use this assay to test if changing the way particular parts of the brain work changes the mouse’s performance. For diseases in which lack of balance and coordination are major features, such as spinocerebellar ataxias, this test is a simple way to check how fast the disease progresses in mouse models. The assay can further be used to test possible treatments for these diseases: better scores after the treatment indicate that the therapy helped the mice improve their balance and coordination.

To sum it up, the balance beam test is a simple and effective assay to measure a mouse’s balance and coordination. Its use helps scientists to understand the basic biology of balance and coordination, as well as uncover why they are impaired in some diseases. Using the balance beam test on mouse models of disease that underwent different treatments, scientists can further measure if the therapy would improve the mouse’s balance and coordination. Therefore, the balance beam test might even help to find new treatments for motor coordination diseases.

If you would like to learn more about the balance beam test, take a look at these resources by the Maze Engineers and Creative Biolabs.

Snapshot written by Dr. Amy Smith-Dijak and edited by Dr.Larissa Nitschke.

Snapshot: What is a Genome-Wide Association Study (GWAS)?

A genome is a person’s complete set of DNA which provides the instructions to make and maintain their body’s functions. Throughout the entire genome, there are genetic differences between individuals known as single nucleotide polymorphisms or SNPs (pronounced “snips”). These variations may be unique or may occur in many people. Normally, these SNPs do not directly cause diseases. But SNPs can sometimes be associated with diseases, and can provide interesting and potentially important information. A genome-wide association study (GWAS) looks at the genomes of many individuals to identify these variations, with the goal of linking more of these variations to particular diseases.

a mural of A, C, T, G repeated over and over again fading off into the distance. It is the genetic code of DNA.
An art piece of repeating A, T, C, and G DNA base pairs, which encode our entire genome. Photo by Stefano on Flickr.

What can these types of studies tell us?

Scientists have gathered plenty of information from GWAS. Once these genetic variations are identified, researchers can use this information to learn more about how diseases occur and affect certain people. For example, GWAS have successfully identified genetic variations that can contribute to diabetes, obesity, and heart disease.

These kinds of studies can also help with creating personalized medicine – where different strategies can be used by doctors to treat patients based on their genetic makeup. This can allow doctors to give patients the most effective treatments, while limiting bad side effects.

How are these kinds of studies conducted?

Researchers typically look at two groups of people: individuals with the disease that is being studied, and people without the disease. DNA is obtained from people in each group to be studied, typically through a blood sample, or skin cells. In order for these studies to work, researchers try to look at as many people as possible. It is a big task, and requires not just hundreds, but thousands of participants! This allows researchers to be confident in the conclusions that they make.

In the early 2000’s, researchers mapped out the complete human genome. Since then, more genetic information from more people have been catalogued. Databases have been created that make it easier for researchers to compare new genomes to ones that have already been sequenced. This makes it quicker and easier to identify genetic variations and how they can contribute to disease.

What has GWAS taught us about SCA?

Some forms of Spinocerebellar ataxia (SCAs) are members of a larger group of diseases known as polyglutamine diseases. This group of diseases are caused by an abnormally long stretch of repetitive segments in the DNA. Scientists have identified that more repeats generally correspond with earlier disease onset, however, this is not always the case. Therefore, scientists have established that disease onset may be affected by other things, such as their environment or other parts of their genome or genetic factors. If researchers can identify these genetic factors, it could improve how these diseases are treated.

The cells in your body are equipped with machinery that helps identify and repair damage to DNA that occurs thousands of times a day from normal cellular processes or the environment (such as sun damage). A few years ago, GWAS revealed that genes involved in these pathways could affect SCA disease onset, and this opened up a new and exciting route of discovery for scientists! Many scientists are currently exploring this route, and more will be done in the coming years to see if we can find new therapies.

If you are interested in reading more about this GWAS report, check out our summary on the paper.

If you would like to learn more about Genome-Wide Association Studies, take a look at these resources by the National Human Genome Research Institute and MedlinePlus.

Snapshot written by Dr. Claudia Hung, edited by Dr. Ray Truant and Celeste Suart.

Snapshot: What is Autophagy?

Autophagy is an important disposal mechanism in our bodies, and it is not as scary as the word sounds. The word autophagy is derived from Greek, with ‘auto’ referring to ‘self’ and ‘phagy’ meaning ‘eating’. Autophagy is important for the growth and development of our cells. It helps to restructure our cells and plays a role in our body’s response to stress, like infection or starvation.

 How does autophagy work?

Autophagy is best compared to a very small recycling depot. Our cells are made of many large and small molecules, also called cellular components. For the sake of our analogy, think of these components as the paper and glue that make up the shipping package from an Amazon delivery. When your Amazon box either is damaged or you no longer have a use for it, you put it in your recycling bin. You will monitor how full or empty it is. When full, it is placed at the curb to be picked up on recycling day.

A high-resolution electron-microscopy image of what an autophagosome looks like in cells. Image from Liza Gross retrieved from Wikimedia.

Your neighbourhood’s recycling pick-up day signals to the city workers what part of the city they need to travel to. The bins outside further specify which houses need their recycling picked up. The contents are taken to the depot, where they are sorted, and the materials can be recycled. Special machinery is used to shred and pulp the cardboard, breaking it down into fibers which are used to make new boxes and other paper products.

When cell components are no longer working properly, like the Amazon box, they must be removed and replaced. Molecules, called protein kinases, monitor how well the cell is working – this is you! Deficits activate these proteins, signaling the need for replacement components and thereby placing your recycling out for pick up. In response to this signal, small pieces of membrane are recruited to an assembly site where they combine to form a half circle – the city workers are here to pick up your recycling! This half circle engulfs the defective cell component(s) and then assembles the rest of the circle to form an autophagosome – your box has finally made it to the depot.

The autophagosome then fuses with the membrane of another storage compartment, called a lysosome, which contains digestive enzymes. These enzymes, like the machinery used to shred cardboard, eat away at the inner membrane of the autophagosome. Once they have worked their way through, they can then target its contents – the cardboard is now being worked into a pulp. The enzymes reduce the damaged goods, inclusive of proteins, into amino acids, sugars, and nucleotides. These products can be either removed from the cell or used for nutrients and energy, just like the processed cardboard fibers reused for other products.

What happens when autophagy does not work?

Looking at spinocerebellar ataxias specifically, some are caused by genes that have a polyglutamine expansion. This type of mutation gives cells the wrong instructions for protein folding. These misfolded proteins huddle together in the brain stem which regulates basic functions like breathing; the cerebellum which coordinates voluntary movements; and the basil ganglia which fine tunes these voluntary movements. When autophagy is working properly, the protein clumps can be cleared away easily. When autophagy is defective, as in spinocerebellar ataxias, the cell cannot get rid of these clusters. The proteins continue to accumulate and are toxic to neurons, contributing to the pathology of neurodegenerative disease.

If you would like to learn more about autophagy, take a look at these resources by the Merriam-Webster and our past Snapshot on Protein Degradation.

Snapshot written by Katie Neuman and edited by Celeste Suart.