By Chris Skeldon 
It has always been thought that associative learning is regulated by the cerebellar cortex, often referred to as the “little brain.” However, new studies resulting from collaboration between… Netherlands Institute of NeuroscienceErasmus MC and the Champalimaud Foundation have shown that cerebellar nuclei contribute in a surprising way to this learning process.
To investigate this question, an international team of researchers in the Netherlands and Portugal, composed of professors Robin Brorsen, Catarina Albergaria, and Daniela Karolyi and with Megan Carey, Katherine Cantu, and Chris Di Zeeu as senior authors, analyzed the mouse cerebellum. With two different stimuli: a short flash of light, followed by a puff of air into the eye. Over time, the mice learned to associate the two stimuli, causing them to protectively close their eyes when they saw the flash of light. This behavioral paradigm has been used for many years as a means of exploring the functioning of the cerebellum.
The center of the production
If we look at the structure of the cerebellum, we distinguish between two main parts: the cerebellar cortex, or the outer layer of the cerebellum, and the cerebellar nucleus, the inner part. These parts are interconnected. Nuclei are groups of brain cells that receive all kinds of information from the cortex, which in turn has connections with other areas of the brain capable of directly controlling muscles, including the eyelid. Thus, the nuclei are essentially the output center of the cerebellum.
According to Robin Brorsen: “The cerebellar cortex has long been considered primarily responsible for learning the eyelid closure reflex and timing. With this study, we show that closing the eyelid at the most appropriate moment can also be regulated by cerebellar nuclei. The teams were working on similar scientific questions, and when we realized the synergy between our studies, we decided to initiate an international collaboration that led to this article.
The cerebellum is influenced by other areas of the brain through various connections, called mossy fibers and climbing fibers. In the experiment described above, it is thought that while mossy fibers carry optical information, climbing fibers convey information relating to air breathing. This information then converges in the cerebellar cortex and nuclei. The Dutch team investigated the effect of associative learning on these connections with nuclei and found that mossy fibers created stronger connections with nuclei in mice that showed associative learning.
Light activation
Meanwhile, the Portuguese team tested the learning ability of the cerebellar nuclei through optogenetics, a method that uses light to control neuronal activity. Katarina Albergaria: Instead of using a normal flash of light to train the animals, we stimulated the brain connections directly, combining that with a puff of air into the eyes. This prompted the mice to close their eyelids at the right moment, showing that cerebellar nuclei could support learning this behavior. To confirm that this learning actually occurs in the nuclei, we repeated the experiments in mice whose cerebellar cortex had been inactivated.
“During learning, the connections between brain cells change,” adds Katherine Cantu. “However, it was not clear where in the cerebellum these changes occurred. We looked at what happens to the input to the cerebellar nuclei from mossy fibers and other inputs during learning and found that in mice that learned — but not in the unlearned mice — the mossy fiber connections to the nucleus became stronger.
Latest technology
“We also observe what happens inside the cell,” Cantu continues, “through electrical measurements made inside the mouse’s nuclear cells.” As is easy to imagine, these cells are very small, about 10 to 20 micrometers. This is smaller than the diameter of a human hair. Using a very thin tube equipped with an electrode, we were able to record intracellular electrical activity while the mouse performed the task, which was a major technical challenge.
“In trained animals, exposure to light caused electrical activity within nucleus cells to change: the cells became more active the closer the air puff got closer in time. Essentially, the cells were prepared for what was about to happen, and so their electrical activity could be precise enough to control In the eyelid before the puffiness occurs.
From mice to humans
“Although this research was conducted in mice, the general anatomy of the cerebellum is similar between mice and humans,” Brorsen points out. Although humans have a larger number of cells, we would expect the connections between cells to be organized in the same way. Our results contribute to a better understanding of how the cerebellum works and what happens during the learning process. This also leads to more knowledge about how damage to the cerebellum affects its function, which may help patients in the future. Activating the connections established with the nuclei, via deep brain stimulation, can make it possible to learn new motor skills.
Megan Carey concludes: “What makes this study unique is the convergence of findings from anatomical, physiological and optogenetic techniques. It is striking how multiple parallel lines of evidence, coming from different teams, are converging to reveal a complete picture of what is happening.”