The regulation of associative learning was traditionally believed to be controlled by the cortex within the cerebellum, commonly known as the “little brain.”
Yet, recent research conducted in collaboration between the Netherlands Institute for Neuroscience, Erasmus MC, and Champalimaud Foundation has unveiled an unexpected role played by the nuclei of the cerebellum in the process of associative learning.
Consider this: If a teacup emits steam, one might wait a little longer before taking a sip. Similarly, if one’s fingers are caught in a door, they are likely to exercise more caution in the future.
These instances represent various forms of associative learning, where positive or negative experiences contribute to the acquisition of behavior. While humans acknowledge the significance of the cerebellum in such learning, the precise mechanisms at play remain a subject of inquiry.
To address this inquiry, an international research team based in the Netherlands and Portugal, comprising Robin Broersen, Catarina Albergaria, Daniela Carulli, and senior authors Megan Carey, Cathrin Canto, and Chris de Zeeuw, examined the cerebellum of mice.
The researchers subjected mice to training involving two distinct stimuli: a brief flash of light followed by a gentle puff of air to the eye.
Over time, the mice acquired the knowledge of an association between these stimuli, prompting them to instinctively close their eyes upon seeing the flash of light. This behavioral paradigm has long served as a method to probe the functioning of the cerebellum.
When examining the cerebellum, it is discernible as having two primary components: the cerebellar cortex, which constitutes the outer layer, and the cerebellar nuclei, located within the inner region.
These components are interconnected, with the nuclei comprising clusters of brain cells that receive diverse information from the cortex. These nuclei subsequently establish connections with other brain regions directly involved in muscle control, such as the muscles responsible for eyelid movement. Fundamentally, the nuclei serve as the output hub of the cerebellum.
The cerebellar cortex has long been regarded as the primary player in learning the reflex and timing of the eyelid closure. With this study, we show that well-timed eyelid closures can also be regulated by the cerebellar nuclei."
Robin Broersen, Researcher, Department of Science, Champalimaud Foundation
Broersen added, “Teams were working on similar research topics and when we realized the synergy of our work, we decided to start an international collaboration resulting in the present article.’
Various connections, known as mossy fibers and climbing fibers, serve as conduits through which other brain regions influence the cerebellum. In the previously detailed experiment, it is hypothesized that the mossy fibers transmit information regarding the light, while the climbing fibers convey information associated with the air puff.
This information subsequently converges within the cortex and nuclei of the cerebellum. The Dutch research team explored the impact of associative learning on these connections leading to the nuclei, discovering that mice exhibiting associative learning displayed enhanced connectivity between the mossy fibers and the nuclei.
Activation With Light
Simultaneously, the team from Portugal employed optogenetics, a technique utilizing light to manipulate cells, to assess the learning capabilities of the cerebellar nuclei.
Instead of using a regular light flash to train mice, we directly stimulated brain connections with light while pairing it with an air puff to the eye. This caused the mice to close their eyelids at the right times, showing that the cerebellar nuclei can support well-timed learning. To ensure this learning was actually happening in the nuclei, we repeated the experiments in mice with an inactivated cerebellar cortex."
Catarina Albergaria, Champalimaud Foundation
Cathrin Canto added, “While learning, connections between brain cells change. Still, it wasn’t clear where in the cerebellum these changes were taking place. We looked at what happens to inputs to the cerebellar nuclei, from the mossy fibers and other inputs, while learning. We found that in mice that learned - but not ones that didn’t - the connections from the mossy fibers to the nuclei became stronger.”
We also visualized what happens inside the cell, by taking electrical measurements inside the nuclear cells of a living mouse. You can imagine that these cells are very small, 10 to 20 µm. That’s smaller than the diameter of a human hair. Using an ultra-thin tube with an electrode, we were able to record the electrical activity inside the cells while the mouse performed the task, an enormous technical challenge."
Cathrin Canto, Erasmus Universiteit Rotterdam
Canto added, “In trained animals, light exposure caused the electrical activity inside the nucleus cells to change: the cells became more active the closer you got to the air puff in terms of timing. Essentially, the cells were prepared for what was to come and could therefore make their electrical activity precise enough to control the eyelid even before the puff had taken place.”
From Mice to Humans
Broersen notes, “Although this research uses mice, the general anatomy of the cerebellum is similar between mice and humans. While humans have many more cells, we expect the connections between cells to be organized in the same way.
Broersen added, “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 functioning, which may help patients in the future. By stimulating the connections to the nuclei using deep brain stimulation, it might be possible to learn new motor skills.”
Broersen, R., et al. (2023) Synaptic mechanisms for associative learning in the cerebellar nuclei. Nature Communications. doi.org/10.1038/s41467-023-43227-w.