Scientists discover unexpected complexity of cerebellar connections

Image of a human Purkinje cell. Almost all Purkinje cells in the human cerebellum have multiple primary dendrites that sprout from the cell body and divide in beautiful, leaf-like patterns. Credit: Silas Busch, University of Chicago

Images of thousands of Purkinje cells reveal that nearly all human cells have multiple primary dendrites. These structures, when observed in mice, facilitate connections with multiple climbing fibers originating in the brainstem.

In 1906, the Spanish researcher Santiago Ramón y Cajal received the Nobel Prize for his pioneering exploration of the microscopic structures of the brain. His famous illustrations of Purkinje cells in the cerebellum depict a forest of neuronal structures, with several large branches sprouting from the cell body and dividing in beautiful, leaf-like patterns.

Despite these early depictions showing multiple dendrites branching from the cell body, the enduring consensus among neuroscientists is that Purkinje cells possess only a single main dendrite that forms a connection with a solitary climbing fiber originating in the brainstem. But a recent study from the University of Chicago, recently published in the journal Sciencereveals that Cajal’s sketches were indeed accurate—virtually all Purkinje cells in the human cerebellum have multiple primary dendrites.

Further studies in mice showed that about 50% of their Purkinje cells also have this more complex structure, and of these cells, 25% receive input from multiple climbing fibers that connect to different primary dendritic branches. Experiments recording cell activity in living mice also revealed that the primary branches can be activated independently and respond to different environmental stimuli.

“The more you work with a particular prototype of a cell in your mind, the more you accept it,” said Christian Hansel, Ph.D., professor of neurobiology at UChicago and senior author of the study, referring to the canonical model that Purkinje cells have one primary dendrite that connects to one climbing fiber. “These drawings by Cajal have been around since the 20th century, so we certainly had enough time to pay attention, but only now with this quantitative analysis can we see that it is almost universal that human cells have several full dendrites each, and we can see that it also makes a qualitative difference.”

Paraphrasing a textbook idea

The cerebellum (from Latin, ‘little brain’) sits at the base of the skull, just above where the spinal cord joins. Ever since the French physician Jean Pierre Flourens first described the function of the cerebellum in 1824, scientists have believed that its sole job was to coordinate movement and muscle activity, but advances in technology have shown that the cerebellum also plays a significant role in processing ​input about the body’s internal and external environment, including sensations of proprioception and balance.

Cerebellar Purkinje cells are like large antennae that receive thousands of inputs that convey a spectrum of contextual information from the rest of the body. These signals are then integrated with a prediction error signal, indicating a mismatch between the context and the brain’s expectation. This error signal is delivered by nerve fibers that climb up from the brainstem and connect to their target Purkinje dendritic structures. Appropriately, these nerves are called “climbing fibers.”

The standard understanding of these connections has been that each Purkinje cell has one primary dendrite that branches from the cell body and connects with one climbing fiber, forming a single computational unit. Belief in this one-to-one relationship between climbing fibers and Purkinje cells, a central dogma of the field that can be found in any neuroscience textbook, comes largely from studies of rodents, which primarily have the single dendrite configuration.

Mouse Purkinje cells. Although 50% of mouse Purkinje cells have a single primary dendrite, the other half have multiple dendrites like human cells. Credit: Silas Busch, University of Chicago

However, many studies of these structures in the past have focused on small numbers of cells, so for this new research, Silas Busch, a PhD student in Hansel’s lab and first author on the paper, started by looking at thousands of cells from both human and mouse tissues. He used a targeted, antibody-based staining technique known as immunohistochemistry to selectively label Purkinje cells in thin slices of the cerebellum. He then categorized the structure of all the cells he could observe and found that more than 95% of human Purkinje cells had multiple primary dendrites, while in mice that number was much closer to half.

“You get a sense of how much this was a prevailing idea in the field because anatomically they are referred to as the ‘primary’ dendrite of a cell,” Busch said. “So even the description of the structure of these cells is based on the mouse prototype that has a dendrite, you can call a primary dendrite.”

This remarkable species difference, in one of the most evolutionarily conserved brain regions shared across mammals and even others vertebrates, led Busch and Hansel to ask whether there might be a functional consequence of having multiple primary dendrites instead of just one. The climbing fiber, with its promising one-to-one relationship and intimate entanglement of the primary dendrite, was their first suspect.

Using sections of mouse cerebellum that contained still-living cells, Busch injected the cells with dye to see their branches and then stimulated climbing fiber input. He observed that 25% of cells with multiple primary dendrites received multiple climbing fibers, rewriting a textbook idea that each Purkinje cell receives only one climbing fiber input, while cells with a single primary dendrite did not.

Walking mice and wiggling whiskers

Encouraged by this finding that a sizable proportion—albeit a minority—of Purkinje cells with multiple primary dendrites also received input from multiple climbing fibers, Busch performed a series of experiments in living mice to see whether this led to functional differences in the living mouse. First, he injected a fluorescent calcium indicator dye into the cerebellum and implanted a small glass window so that he could later observe the flow of calcium into the Purkinje cell dendrites. By holding the mouse’s head under a microscope while it ran on a treadmill, he could measure calcium flow that indicated when a climbing fiber is providing a strong input to the cell. In cells with one primary dendrite, high-resolution images showed that the activity signal was uniform across its structure; in cells with multiple primary dendrites, he could detect activity on each side at different times, meaning that one dendrite could be activated by its climbing fiber while the other dendrite in the same cell was not.

Next, Busch wanted to see if he could tease out individual climbing fiber activity using a more precise stimulus: the mouse’s whiskers. For this experiment, however, Busch had to sedate the mice (“I don’t know if you’ve ever tried to stimulate individual whiskers in an awake mouse, but it’s really hard,” he said). While the mice slept, Busch inserted individual whiskers into a small glass tube and wiggled them back and forth. Here he could also see activity in separate dendritic branches of the Purkinje cells, suggesting that individual climbing fibers signaled input from individual whiskers to individual dendrites.

Finally, for a more real-world scenario, Busch also tested awake mice with several stimuli, such as flashes of light, sounds, or puffs of air on the whisker pad. Again, he saw differences across the Purkinje cells. In some, one branch will differentially favor one stimulus, so that it may be particularly responsive to light but not sound. Then the other branch may respond preferentially to sound, but not light.

“This happened in a minority of cells, since there are fewer with more branches in mice, and not all of them get more climbing fibers, but still the presence of this effect was very interesting,” Busch said. “It confirmed this idea that the two climbing fiber inputs will have different functional purposes, representing different information.”

The cerebellum connection becomes more apparent

This new evidence overturns standard thinking about a brain region thought to be reasonably loose anatomically and also having functional implications. As the climbing fibers provide input from the brainstem, the Purkinje cells assemble and process this information. Multiple inputs that connect at multiple points on the cells provide more computational power, allowing brain circuits to adapt and respond to changes in the environment or the body that require different movements, and this non-canonical connection is closely related to the structure of Purkinje cell dendrites.

There is also evidence that these connections in the cerebellum may be involved in disease. In 2013, for example, Hansel worked on a study with UChicago neurologist Christopher Gomez, MD, Ph.D., that showed that Purkinje climbing fiber connections are weaker in mouse models of cerebellar ataxia, a movement disorder. On the other hand, Busch, Hansel and Gomez have published work with former UChicago student Dana Simmons showing that these connections are stronger in genetic duplication and overexpression models of autism. Other researchers also show stronger connections in certain types of tremors. Understanding more about the essential biological structures of these cells will hopefully provide more insight into these relationships.

“People who study other parts of the brain like the neocortex or the hippocampus always have more or less an idea of ​​what that brain structure does,” Hansel said. “Those of us who study the cerebellum always had the idea that it is motor coordination and adaptation, but it was also clear that it was something beyond that. Now it becomes easier to understand as the connection becomes clearer.”

Reference: “Climbing fiber multi-innervation of mouse Purkinje dendrites with arborization common to humans” by Silas E. Busch and Christian Hansel, 27 July 2023, Science.
DOI: 10.1126/science.adi1024

The study was funded by the National Institute of Neurological Disorders and Stroke, the National Institute of Neurological Disorders and Stroke, and University of Chicago Pritzker Fellowship.