Serotonin, a brain chemical widely recognized for its influence on mood and anxiety, has been found to operate in a surprising way within the cerebellum, according to new research published in The Journal of Neuroscience. Scientists discovered that increasing serotonin levels in this specific brain region of mice actually lessened anxiety, an effect opposite to what is often observed elsewhere in the brain. This finding highlights a novel mechanism by which the cerebellum contributes to anxiety regulation.

Scientists have long been interested in unraveling the complex brain circuits that govern anxiety. A better understanding of these circuits could pave the way for more targeted and effective treatments for anxiety disorders, which affect millions of people worldwide. Previous research had separately pointed to both serotonin, a neurotransmitter known to influence mood, and the cerebellum, a brain region located at the back of the head, as playing potential roles in anxiety.

Serotonin is well-established as a key player in anxiety regulation in other brain areas, and medications like selective serotonin reuptake inhibitors, which increase serotonin levels, are commonly used to treat anxiety. Similarly, clinical observations and studies in animals had suggested a connection between the cerebellum and anxiety, but the precise nature of this link remained unclear.

Researchers Pei Wern Chin, from the University of Pennsylvania, and George Augustine, from Temasek Life Sciences Laboratory, sought to investigate whether serotonin within the cerebellum itself directly influences anxiety behavior. They aimed to explore if manipulating serotonin levels in this brain region could have a measurable impact on anxiety.

“I have been studying the cerebellum for more than 30 years. The attraction is its relatively simple, stereotyped circuitry. If we can ever hope to figure out any part of the brain, the cerebellum is a prime candidate,” explained Augustine, a Temasek Senior Investigator and co-editor of the textbook Neuroscience.

“Pei Wern was interested in anxiety. So we joined forces to look at the role of the cerebellum in anxiety. Originally, she identified a part of the cerebellum (lobule VII) that serves as a locus for anxiety regulation. Serotonin is widely known to be involved in anxiety, so it was a logical next step to examine the role of serotonin in cerebellar anxiety regulation. That was the motivation for our current paper.”

To explore this, the researchers conducted a series of experiments using mice. First, they wanted to measure the natural levels of serotonin in the cerebellum of mice exhibiting different anxiety levels. They used a sophisticated tool: a fluorescent sensor specifically designed to detect serotonin. This sensor, called GRAB5HT2h, was introduced into a specific part of the cerebellum called lobule VII. This sensor glows brighter when it detects serotonin, allowing the researchers to monitor serotonin levels in real-time.

To get the sensor into the cerebellum, they used a harmless virus to deliver the genetic instructions for making the sensor to the brain cells in lobule VII. After allowing time for the mice to produce the serotonin sensor, the researchers made thin slices of cerebellar tissue and examined them under a special microscope. They applied serotonin to these slices and confirmed that the sensor effectively responded to serotonin by glowing more brightly, demonstrating its ability to detect serotonin in the cerebellum.

Next, the scientists wanted to measure serotonin levels in the cerebellum of living, behaving mice. They implanted a small optical fiber into lobule VII of the mice that had the serotonin sensor. This fiber allowed them to shine light into the cerebellum and measure the fluorescence signal from the serotonin sensor, a technique called fiber photometry. They also implanted a tiny needle into a nearby cerebellar region to inject substances directly into the cerebellum.

To ensure the sensor was indeed detecting serotonin in living mice, they injected serotonin into the cerebellum through the implanted needle and observed an increase in the sensor’s fluorescence. Conversely, they injected a substance that blocks serotonin receptors and saw a decrease in the sensor’s signal. These tests confirmed that the sensor was accurately reporting serotonin levels in the cerebellum of live mice.

With the serotonin sensor in place, the researchers then assessed the anxiety levels of the mice using a standard behavioral test called the elevated zero maze. This maze is a circular track raised above the ground, with two open sections and two enclosed sections. Mice naturally prefer enclosed, safer spaces and tend to avoid open, exposed areas, reflecting their anxiety levels.

The researchers recorded the behavior of the mice on the maze while simultaneously monitoring serotonin levels in their cerebellum using fiber photometry. They found a striking pattern: mice that spent more time in the open, anxiety-provoking sections of the maze had higher levels of serotonin in their cerebellum. Conversely, mice that exhibited more anxiety-like behavior, spending more time in the enclosed sections, had lower levels of cerebellar serotonin. This inverse relationship was a key initial finding.

Building upon this observation, the researchers then sought to directly manipulate serotonin input to the cerebellum and see if it would change anxiety behavior. They used a technique called optogenetics, which allows for the precise control of brain cell activity using light. They genetically modified a group of mice so that their serotonin-releasing neurons could be activated or inhibited by light.

In one experiment, they used light to stimulate serotonin release in the cerebellum while the mice were on the elevated zero maze. They found that stimulating serotonin release caused the mice to spend significantly more time in the open sections of the maze, indicating reduced anxiety.

“In the past, the cerebellum was supposed to be involved just in motor coordination,” Augustine told PsyPost. “Our paper adds to the growing evidence that the cerebellum is involved in cognition, too. Serotonin, which is known to regulate anxiety by acting on other brain areas, also does so in the cerebellum.”

In another experiment, they used light to inhibit serotonin release in the cerebellum. This time, they observed the opposite effect: the mice spent less time in the open sections and more time in the closed sections, showing increased anxiety. These optogenetic experiments provided strong evidence that serotonin in the cerebellum has a direct and causal role in regulating anxiety behavior. By increasing serotonin, they could reduce anxiety, and by decreasing serotonin, they could increase anxiety.

“The big surprise is that in most brain areas, serotonin increases anxiety,” Augustine explained. “However, in the cerebellum it is the opposite: increasing serotonin levels reduces anxiety! That is why we can think of the cerebellum as a ‘brake’ on anxiety.”

While these findings are significant, the researchers also acknowledged some limitations. This study was conducted in mice, and it is important to determine if these findings translate to humans. “Mice are great models for many types of neuroscience work, especially because we can apply genetic engineering (which we took advantage of in our work),” Augustine noted. “However, while our brains also have a cerebellum, definitely further work will be needed to see whether our conclusions apply to the human brain.”

Future research could investigate the precise neural circuits involved, exploring how the cerebellum interacts with other brain regions known to be involved in anxiety, such as the amygdala. One important question for future studies is to identify the specific neurons that supply serotonin to the cerebellum and determine if these are the same neurons that send serotonin to other brain regions that influence anxiety. Understanding the broader brain circuitry will provide a more complete picture of the cerebellum’s role in anxiety.

“The long-term goal in mice would be to complete the circuit: filling in the missing pieces of the puzzle by identifying the precise relationship between the cerebellum and the other parts of the brain involved in anxiety behavior,” Augustine said

“I would like to emphasize that Pei Wern Chin was the mastermind of this project,” he added. “She came up with most of the good ideas and definitely did 100% of the very arduous experimental work.”

The study, “Serotonergic Input into the Cerebellar Cortex Modulates Anxiety-Like Behavior,” was published February 10, 2025.