Learning to avoid a threat as well as knowing when something is no longer dangerous is crucial for an organism’s survival. Being able to shake off fearful memories after a threat has passed is so important that deficits are linked to several neuropsychiatric conditions such as anxiety and post-traumatic stress disorder (PTSD). Over the last decade, evidence has shown that changes in the gastrointestinal (GI) microbial community, collectively known as gut microbiome, influences brain-related processes such as fear learning and memory. But how these invisible creatures could do that remained unclear until a few years ago.
“…the gastrointestinal (GI) microbial community, collectively known as gut microbiome, influences brain-related processes such as fear learning”
In a study published in 2019, researchers investigated how changes in an animal’s GI microbiota would affect neuronal function and their ability to extinguish a fear memory (Chu et al., 2019). The authors trained germ-free (i.e. animals with no microbiome at all), antibiotic-treated (i.e. animals with dramatically reduced microbiome), and control (i.e. animals with a fully-functioning microbiome) mice to associate a loud noise with an electric foot shock. This learning process – in which an animal learns to associate stimuli – is called classical or Pavlovian fear conditioning, and many researchers use it to understand how fear memories are acquired, formed, and maintained in the brain. Before fear conditioning, the loud noise is called a neutral stimulus, while the electric foot shock is known as unconditioned stimulus. After fear conditioning, the sound becomes a conditioned stimulus, as it induces behavioral and/or physiological changes known as conditioned responses. These are reflex responses (i.e. involuntary responses) that range from behavioral modifications, such as expression of defensive responses (e.g. freezing behavior), to physiological changes such as increased heart rate.
After Chu and colleagues conditioned mice to associate the sound with an electric foot shock, they repeatedly exposed the animals to the noise in the absence of the shock. This procedure gradually decreases the expression of conditioned responses through a learning process known as fear extinction. Evidence indicates that fear extinction represents new learning in which an animal learns to disassociate the loud noise from the electric foot shock. In turn, this new association (i.e. noise-no-shock) inhibits the expression of the conditioned responses. In Chu and colleagues’ study, microbiome depleted animals (i.e. antibiotic-treated and germ-free mice) and control mice had similar levels of freezing behavior during fear conditioning, suggesting that these groups similarly acquired the noise-shock association. However, during extinction, microbiome depleted mice did not extinguish the fear memory as well as control animals, indicating that microbiota may be important to shake off fearful memories.
“Evidence indicates that fear extinction represents new learning”
Next, the researchers investigated the genetic and neuroanatomical underpinnings of these changes. They focused their attention on the medial prefrontal cortex (mPFC), a brain region located in the front of the brain that has been implicated in fear extinction. Researchers found that within the mPFC, genes related to several functions such as synapse plasticity and neuronal activity were less expressed in animals with depleted microbiomes. Besides regulating how genes are expressed in the mPFC, the gut microbiome also affected the development and morphology of cells in that region. Microglia cells, which are important for maintaining neuronal function, exhibited an immature state in animals with compromised microbiomes. Moreover, these animals also showed changes in dendritic spines. These are small membrane protrusions present on the surface of a neuron’s dendrite that are crucial for synaptic function and are known to be remodeled by learning. Mice with impoverished microbiomes had fewer dendritic spines that were eliminated more often, indicating that depletion of the microbiome negatively impacts learning-related spine plasticity. Together, these results showed that reduction of the gut microbiota not only alters the behavioral expression of fear extinction, but also modified the expression of genes, development, and morphology of cells in the mPFC.
“Mice with impoverished microbiomes had fewer dendritic spines“
But how do microbes living in the gut cause these changes in the brain? To determine the mechanisms mediating these changes, researchers tested how microbes were sending signals to the brain. First, they explored the theory that the signals were sent through the vagus nerve, which connects the GI tract and the brain. They found that snipping the vagus nerve did not alter animals’ ability to extinguish the fear memory. Then, they tested if changes in fear extinction were mediated by changes in the immune systems, given that immune cells are known to influence brain function and behavior. Again, the authors did not find differences in the number of immune cells among the groups. Finally, they assessed whether metabolites produced by the microbes were mediating such changes. They discovered that four compounds named Phenyl sulfate, Pyrocatechol sulfate, 3-(3-sulfooxyphenyl) propanoic acid, and Indoxyl sulfate were reduced in the cerebrospinal fluid, blood serum, and stool of germ-free mice. Interestingly, indoxyl sulfate and a derivative of 3-(3-sulfooxyphenyl) propanoic acid were previously linked to psychiatric conditions such as schizophrenia and autism in humans. Together, these findings indicate that microbiome-derived substances make their way to the brain and influence the activity of brain cells that, in turn, lead to changes in how well mice extinguished the fear memory.
“They discovered that four compounds […] were reduced in the cerebrospinal fluid, blood serum, and stool of germ-free mice”
These interesting findings indicate that an animal’s ability to extinguish fear is not only dependent on changes inside its brain, and bring the invisible inhabitants of the gut to the spotlight as key modulators of that process. This and other studies shed a light on how gut microbes influence the brain, and it might pave the way to a better understanding of neuropsychiatric disorders like PTSD, which have fear memory alterations as a hallmark.
Written by Mariella Careaga.
Edited by Elizabeth Burnette, Arielle Hogan and Desislava Nesheva.
Illustrated by Melis Cakar.
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Chu, C., Murdock, M. H., Jing, D., Won, T. H., Chung, H., Kressel, A. M., … & Bessman, N. J. (2019). The microbiota regulate neuronal function and fear extinction learning. Nature, 574 (7779), 543-548.
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Sierra-Mercado, D., Padilla-Coreano, N., & Quirk, G. J. (2011). Dissociable roles of prelimbic and infralimbic cortices, ventral hippocampus, and basolateral amygdala in the expression and extinction of conditioned fear. Neuropsychopharmacology, 36(2), 529-538.
VanElzakker, M. B., Dahlgren, M. K., Davis, F. C., Dubois, S. & Shin, L. M. (2014). From Pavlov to PTSD: the extinction of conditioned fear in rodents, humans, and anxiety disorders. Neurobiology of Learning and Memory, 113, 3–18.