Are you a socially adept or a socially phobic person? Let your intestines decide.

In recent decades, scientists have come to understand that the gut and brain have a powerful interplay. For example, certain types of intestinal ulcers have been linked to worsening symptoms in Parkinson's patients. Clinicians have also long known that gastrointestinal disorders are more common in patients with neurodevelopmental disorders such as attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD).

" Not only does the brain influence the gut, but the gut can also profoundly influence the brain ," says Kara Margolis, a pediatric gastroenterologist at New York University's Langone Health. However, exactly how these two anatomically separate organs influence each other is unclear.

In a paper published in PLOS Biology in early November this year, researchers found that zebrafish lacking intestinal microbiota were much less social than zebrafish with intestinal microbiota, and their brain structure also reflected this difference. Another related article published in BMC Genomics in late September this year also described the molecular characteristics of neurons affected by intestinal flora.

Philip Washbourne, a molecular biologist at the University of Oregon and one of the lead researchers on the new study, has spent the past two decades studying genes involved in autism and the development of social behavior. He and his lab team were looking for a new model organism that would exhibit social behavior but would be quicker and easier to breed than the first choice: mice. “Can we do this in fish?” he recalls thinking, then saying, “Let’s try to quantify it in fish and see if we can have some parameter to measure how friendly fish are.”

The story of sterile fish

Zebrafish are already widely used in genetic research, they reproduce quickly and are naturally social creatures, starting to swim in groups of 4 to 12 after two weeks of age. Zebrafish are transparent until adulthood, so researchers can observe their internal development without dissecting them , which is simply impossible in mammalian models such as mice.

The embryos used by the research team for the experiment came from "sterile" zebrafish that lacked a gut microbiome. After the fish hatched, the researchers immediately inoculated some of them with a healthy mixture of gut bacteria. The remaining fish were inoculated a week later, so these fish were in a "sterile" stage in the early stages of development.

Zebrafish that were inoculated with microbes at birth began to group together as expected at about 15 days of age, while those that began their development in a sterile state " shockingly did not group together ," said Judith Eisen, a neuroscientist at the University of Oregon and one of the authors of the study. Even when these fish were subsequently inoculated with gut microbes, they still could not achieve the same degree of sociality as their peers.

Eisen, Washburn and their team studied the brains of these fish and found significant differences in brain structure between the two groups of fish. Those zebrafish that were "sterile" in the first week of life showed more interconnections between a cluster of forebrain neurons, which affect the social behavior of fish . At the same time, there was a significant decrease in microglia in this cluster of cells. Microglia are a type of neuro-immune cell that is responsible for cleaning up garbage in the brain. Eisen said, "These are major changes in the nervous system."

The lines show the paths that zebrafish swim in special experimental tanks. Fish raised under normal microbiome conditions (above) spend more time near the transparent partition in the tank to get close to the fish on the other side of the partition. Fish raised under "sterile" conditions (below) are less sociable and swim more randomly. (Image source: original paper)

The team hypothesizes that healthy gut microbes somehow encourage microglia to flourish in the zebrafish brain. Then, at certain critical periods of development, these microglia act like gardeners, pruning the wildly branching "arms" on neurons. Without microglia pruning these branches, the social neurons in the fish's brains become tangled and overgrown , like untended bushes. It's not clear how gut microbes send signals to the developing brain to produce these effects. Bacteria can release a large variety of chemicals, and in theory any compound small enough can cross the blood-brain barrier. However, it's also possible that immune cells carry signaling molecules as they travel between the gut and the brain, or that some signals travel from the gut to the brain along the vagus nerve.

Gut microbes in other social animals

The same mechanism may also be at work in other vertebrates, including humans. Socializing is a common survival strategy in the animal world. "It's a behavior that's been conserved during evolution," said Livia Hecke Morais, a biologist at the California Institute of Technology.

In an article published in Frontiers in Microbiology in November this year, researchers collected fecal samples from a rhesus monkey group on Santiago Island off the coast of Puerto Rico, and used the length of time spent grooming each other and the number of partners as indicators of social ability. The study found that some bacteria that are beneficial to host immunity (such as Faecalibacterium and Prevotella) are more abundant in the feces of rhesus monkeys with stronger social skills , while the typical pathogenic bacteria, Streptococcus, are more abundant in the feces of rhesus monkeys that are not good at socializing. At the same time, some intestinal bacterial genera that are reduced in human autism patients are also less in unsocial macaques.

However, whether sociability affects the macaques' gut microbiota, or the gut microbiota affects sociability in turn, or whether the two influence each other, is not clarified in this article. But Washburn and Eisen previously found social neurons in mice that are almost identical to those in zebrafish, which may mean that the influence of zebrafish gut microbiota on sociability may also occur in mammals in the same way. Washburn said, "If you can find the same type of cells in fish and mice, then you are likely to find the same cell type in humans."

View image of Zebrafish typically begin socializing in schools when they are about two weeks old (Credit: Lynn Ketchum/University of Oregon)

However, Morais cautions that neither zebrafish nor mice are perfect analogs for humans . The neural pathways in fish and mice are a little different, she says, and the species have their own distinct gut microbiomes that may release different chemical signals. Still, the principle is likely to apply broadly to different groups of organisms. It’s still possible that chemicals produced by different microbes could affect the number of microglia in the brains of zebrafish, mice, humans or other animals, Eisen says. But she agrees that it’s dangerous to lump different species together, and that model organisms “are not exactly the same as humans.”

Diversity of the gut microbiota

In the future, Eisen, Washburn and their research team hope to find out exactly how the gut microbes in zebrafish send signals to the brain. They also want to determine how long the sensitive period of neurodevelopment is to understand whether early intervention in the gut can get brain development back on track. Ultimately, they hope that this research will lead to a deeper understanding of how neurodevelopmental disorders arise in the human population, however difficult it may be.

“The problem is that this hypothesis has to be tested in humans ,” Margolis says, “and that’s challenging to do.” Designing a clinical trial of a gut intervention in human infants would be difficult because conditions such as autism spectrum disorder are often not diagnosed until children are 7 or older, which is likely long after the critical window for interventions in young children to have an impact has closed.

However, a clinical trial conducted in 2017 in 18 children with autism spectrum disorder aged 7 to 16 showed that after 10 weeks of microbiota transfer therapy, a certain dose of standardized human intestinal microbiota was given orally or rectally, and the children's autism spectrum disorder-related behaviors were significantly improved, and there was no reversal of the disease within 8 weeks of the end of treatment. This may suggest that the window period for this intervention is longer than expected.

The standardized human gut microbiota used in this clinical trial was prepared from the feces of healthy individuals, but even the same species can vary significantly between individuals. Two people who look exactly the same in every way can have gut microbiotas that differ by more than 70%. Looking at an individual's microbiota alone is not enough to diagnose a neurodevelopmental disorder, Margolis said. " There is no one microbiome that is an autism microbiome ." For Washburn, if humans do have such a developmental period of sensitivity to gut microbes, then intervening in it is almost impossible. He said, "I don't think we're any closer to a so-called panacea." However, even if we can describe in some small way the impact of the gut on the brain, it will help to solve this extremely complex human mystery. For now, he said, this is enough.