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As of 2010, anxiety has become the sixth most common disability worldwide, impairing the lives of millions. This stress-induced condition steadily increased during the pandemic, sparking the interest of mental health researchers. While environmental stresses may worsen anxiety, genes play a role, too.

At the University of California, a team of researchers were interested in how turning genes on or off affects anxiety symptoms. The team began to decipher the problem by conducting mouse experiments on the gut microbiome, a community of microorganisms that live in the intestines of mammals, including humans, and help us digest and absorb nutrients.

In previous studies, researchers have identified many genes that are associated with anxiety-like behaviors, making it possible to sort individuals with low and high anxiety based on these genetic characteristics. We also know that there are distinct groups of bacteria living in our gut that are more common in people with anxiety-like behaviors. In their background reading, the researchers learned that your gut microbiome might even predict what neurological diseases you’re susceptible to.

The researchers at UC wanted to understand the relationship between certain behaviors and bacterial strains, so they used specially bred mice that have a similar amount of genetic variation to humans. These lab subjects are known as cross-collaborative mice, or CC mice. Their genetic variation is key to helping researchers understand not only microbiome composition, but also cancer susceptibility and memory performance in humans. According to data from the UNC Systems Genetics Core, where the mice in the UC study were purchased, these mice had 264 sequences that matched 141 known sequences that other scientists have found to be associated with anxiety-like behavior across multiple species.

Researchers wanted to first evaluate anxious behavior in CC mice to determine if their behavior was associated with a particular microbiome composition. This was done through measuring stress responses in the animals through what is known as a light/dark test. This experimental device exposed 445 CC mice, which represented 30 genetic variations, to lighted compartments followed by completely dark compartments. 

Using video, the scientists observed and recorded the number of times the mice transitioned to the lighted compartment, the speed in which they traveled, and how long they stayed there. Excessively avoiding light is considered to be an anxious behavior in mice. Measuring these activities allowed the scientists to identify anxiety-related mannerisms among the mice and see if particular genetic groups had more or fewer of these behaviors.

Once the behavioral characteristics of the mice were identified and measured, the mouse data was divided into “low” and “high” anxiety groups. Fecal samples had been collected from the mice 24 hours before the experiment, which the researchers used to get a baseline reading of the gut bacteria. The samples underwent DNA extraction using a lab kit called the PowerSoil DNA Isolation Kit. The recovered DNA was then sequenced using a standard protocol called 16S rRNA sequencing on a device called a MiSeq, which sequenced only the bacterial DNA. These tools allowed the researchers to identify all of the bacterial families living in the guts of the different CC mice.

To determine if there is, in fact, a multi-layered relationship between anxiety behaviors, genetics, and gut bacteria, the researchers used statistical software, known as ConsensusClusterPlus to pinpoint the genetic variants that were most prevalent among mice with high anxiety behaviors. The software uses a technique known as consensus clustering, which is a computer algorithm that groups genes based on their similarity. This allowed researchers to see relationships between behavior, bacterial genes, and the mouse genes.

The bacterial genus Ruminococcaceae, as well as the genera of Clostridiaceae and Clostridiales, were all notably higher in high anxiety mice than in low anxiety mice. These bacterial groups were found less often for mice that spent more time in the light box. They also found seven locations on the mice’s DNA which were positively correlated with gut bacteria known to cause inflammation, a symptom related to stress. The researchers say these data suggest that the gut microbiome may be involved in the regulation of anxiety-like behaviors.  

There is still much to be understood about this intricate and symbiotic relationship between the gut microbiome, genetics, and behavior. Studying the brain-gut connection is only the beginning of accurately understanding the origins of psychological disorders. Environmental factors that contribute to anxiety will always exist, however, the further these studies go, the closer we get to a more well-rounded approach to helping those suffering from mental health conditions.

Study Information

Original study: Gut microbiome partially mediates and coordinates the effects of genetics on anxiety‑like behavior in Collaborative Cross mice

Study published on: 11 January 2021

Study author(s): X. Jin, Y. Zhang, S.E. Celniker, Y. Xia, J.-H. Mao, A.M. Snijders and H. Chang

The study was done at: University of California (USA)

The study was funded by: Lawrence Berkeley National Laboratory Directed Research and Development (LDRD)

Raw data availability: Author statement: "All data needed to evaluate the conclusions in the paper are presented in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Mouse gut microbiome 16S rRNA gene sequence data is available on OSF."

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