Thesis Watch: Isaac Van Flein, Biological Sciences
By Zeke Shomler
On Tuesday, October 10th, I got the chance to sit in on Isaac Van Flein’s M.S. thesis defense in Biological Sciences. His research, titled “Effects of Desformylflustrabromine on Compulsive-like and Social Behavior in Mouse Models of OCD and Autism,” is focused on behavioral effects of a potential treatment for several adverse symptoms of Obsessive-Compulsive Disorder (OCD) and Autism Spectrum Disorder (ASD).
OCD and ASD, as Isaac explained during his presentation, frequently co-occur and can involve similar symptoms. They can, for example, both present with repetitive, restricted, and compulsive behavior patterns. These have been linked in both OCD and ASD to a part of the brain called the “cholinergic system.” Although, actually, to call it a “part of the brain” is a bit of a misnomer, since the cholinergic system spreads across multiple brain regions and throughout the body in the peripheral nervous system. “Cholinergic” means this system involves the production and uptake of a neurotransmitter called acetylcholine, which is vital for many human functions, including learning, memory, sensory processing, and behavioral regulation.
One particular acetylcholine receptor in this system, called the α4β2 (that’s “alpha-four-beta-two”) nicotinic acetylcholine receptor, has been linked with a new potential treatment, desformylflustrabromine (dFBr). dFBr hangs out on the side of the α4β2 receptor and makes it much more sensitive to the critical behavior-moderating neurotransmitter, acetylcholine. In the past, treatment with dFBr has been seen to reduce depressive and compulsive-like behaviors in mouse models of OCD and ASD.
Mouse models are an important part of biological and medical research. They allow us to see the behavioral and physiological effects of potential treatments without putting human patients at risk. We understand their brains very well, and the fact that they’re mammals, specifically euarchontoglires, means aspects of their biology are very similar to ours. They also grow and reproduce quickly enough to make research projects feasible. As Isaac stated during his presentation, “Animal models are invaluable biomedical research tools.”
Isaac did a lot of work alongside his advisor, Dr. Abel Bult-Ito, to manage and study the mouse models for this research. The OCD mouse model was developed by Dr. Bult-Ito through a selection of over fifty-five generations for compulsive behaviors, as indicated by nest-building habits. In short, mice were bred that have a compulsive tendency to create very large nests—to keep repetitively adding nesting material long after they’ve created a sufficient place to rest and raise their young. Compulsive behaviors, Isaac explained, are essentially ordinary behaviors performed excessively to the point that they disrupt the patient’s life, often to relieve related obsessive thoughts and anxieties. In humans, these can become problematic when they interfere with the ability to perform basic functions. It is known to us as OCD.
But how well can mice actually replicate something as complicated as OCD or autism? That question, Isaac explained, is something that scientists have thought about a lot. Although we can’t ask the mice how they feel directly, we can determine the accuracy of a model by looking at a combination of three things: its superficial resemblance in behavior patterns to human behaviors, its “predictive validity,” or similar response to drugs and therapies, and its “construct validity,” or having similar biomarkers and physiological effects. Dr. Bult-Ito’s model, which is often used in behavioral research here at UAF, is particularly good across all three categories. The mice that were bred to model OCD are called the “HA strain.” These were compared to a non-compulsive “LA strain.”
An HA litter shortly before weaning
Photo by Isaac Van Flein
Isaac also worked to model Autism Spectrum Disorder in mice. To do this, he (very carefully) added valproic acid to fetal mice’s brains after about twelve days of gestation. Valproic acid interferes with cortical development and produces behavioral patterns that resemble the symptoms of ASD. By treating mice with valproic acid in both the HA and LA strains, he had groups representing OCD, ASD, and comorbid OCD/ASD conditions, in addition to the non-treated, non-compulsive LA control. While valproic acid has been used extensively for studying the neurochemistry of ASD, comorbid models are less common and Isaac is the first to attempt to convert Dr. Bult-Ito’s OCD mouse model into a comorbid OCD/ASD model.
Across all these different groups, Isaac treated some mice with dFBr, and others with saline to serve as a control group. Then, between Summer 2021 and Summer 2022, he meticulously put them through five different tests to see how the treatment would affect their behavior.
The first test, called the “nest building test,” produced data about the mice’s tendency toward compulsive behavior. They were put into a cage with unlimited cotton (some of their favorite nest-building material), and the size of their nests were measured after one, two, three, four, five, and then twenty-four hours. The building of excessively large nests and continuation of nest-building activity for a long time both pointed toward compulsive behavior patterns.
An LA mouse after completion of the nest building test
Photo by Isaac Van Flein
For the second test, the “marble burying test,” mice were put into a bin filled with bedding material that had a grid of marbles on top. They were then given twenty minutes to dig around in the bedding. The more the mice moved around, the more the marbles would move toward the bottom of the bin. Greater movement was associated with anxiety and with compulsive behaviors.
The third test, the “open field test,” measures the movement of the mice in an open square field divided into an inner square section and an outer zone. Greater time in the outer section indicated “wall-hugging” behavior that is indicative of anxiety, and a greater number of crosses between the two zones indicated confidence and willingness to explore.
The fourth test is often used to measure depressive-like symptoms. It’s called the “tail suspension” test, and is pretty straightforward: hold the mouse by its tail for ten minutes and measure how much it wiggles around. The more time a mouse spends still when held in that position, the more it mimics depressive behavioral symptoms.
These first four tests are commonly used in behavioral research with mice. The fifth test is also commonly used to study social behavior, but has never been applied to Dr. Bult-Ito’s OCD mouse model. Called the “three-chamber social test,” it helped him measure sociability and the mice’s aptness to seek novel social stimulation.
The mice were first put into the center of a container with three separate sections. For ten minutes, it was left alone, and Isaac measured how much they moved around. Then, a “social stimulus mouse” was put into one of the side chambers, and he monitored how much time the test mouse spent both in the chamber and up close to the new mouse. Then, after another ten minutes, a novel mouse was put into the third chamber, and again, he measured how much time the test mouse spent socializing with the novel mouse, with the first mouse, or neither. This new test is especially useful for research related to potential social deficits associated with ASD.
By the end of these behavioral tests, conducted across multiple generations of mice in all four combinations of drug/saline treatments in both the HA and LA strains, Isaac had collected, in Dr. Bult-Ito’s words, “a massive amount of data.” Isaac then spent another year analyzing it and preparing it for publication.
He ultimately found that, surprisingly, the dFBr did not have a significant effect on the compulsive or depressive behaviors, as has been shown in previous similar studies. He explained that might have been from the chemical staying in the refrigerator too long, or being otherwise compromised. Either way, it provides valuable data toward the ongoing question of possible treatment for OCD symptoms.
The ASD mouse model didn’t quite go to plan, either—probably because the dosage of valproic acid was too low. A previous study, Isaac explained, had performed tests that suggested a lower-than-usual dose could replicate the behavioral symptoms with lower offspring mortality rates, but another study came out during Isaac’s research concluding that the dose suggested by the first researcher was a bit too low to provide a usable behavioral model. So, although Isaac didn’t successfully replicate ASD symptoms in his mice, he did contribute to an important body of research about valproic acid, confirming the need for a slightly higher dose. His effort to reduce the infant mortality rate of his test subjects is commendable, and his research provides data that will help future researchers get a more balanced experimental dosage.
Future research in this field of study will likely involve re-attempting the valproic acid treatment, and further studying the acetylcholine levels in the OCD mouse model. dFBr can also be tested on other mouse models, such as wild-type mice and alternative ASD models. As Isaac stated during his defense, “I still think dFBr has a lot of potential as a therapeutic treatment.”
Isaac earned his B.S. in Biological Sciences here at UAF, studying the same OCD mouse model strain for an undergraduate thesis project. After graduation, he hopes to use his biological knowledge and research skills to work for the state. In his words, he hopes for a job “where I can make a difference managing wildlife or working to address climate change, something that makes an impact and has value with regard to these enormous ecological issues facing human civilization.”
He also has some valuable advice when it comes to academics: “You can go at your own pace. Trying to adhere to arbitrary standards of when and how fast you should get a degree was just stressful and distracting.”
Isaac also hopes to continue his current work as a science communicator to get more people interested in science. There is still much research to be done about the cholinergic system and its effect on behavioral manifestations of OCD and ASD. As he states, “this is an ongoing area of research with much to be learned!”