Human Purkinje cell image. Almost every Purkinje cell in the human cerebellum displays numerous primary dendrites branching from the cell body and breaking into stunning, foliate patterns. Credit: Silas Busch, University of Chicago
Visualizations of thousands of Purkinje cells expose the fact that virtually all human cells contain multiple primary dendrites. In mice, these structures enable connections with various climbing fibers that originate from the brain stem.
Santiago Ramón y Cajal, a Spanish researcher who received the Nobel Prize in 1906 for his pioneering investigation of the brain’s microscopic structures, is well known for his detailed depictions of Purkinje cells within the cerebellum. His drawings present a myriad of neuronal structures, including numerous large branches radiating from the cell body and forming magnificent leaf-like patterns.
Despite early depictions illustrating several dendrites branching from the cell body, it has been widely accepted in neuroscience that Purkinje cells contain a single major dendrite which connects with one climbing fiber from the brain stem. However, a recent study from the University of Chicago, recently featured in the Science journal, confirms the accuracy of Cajal’s sketches — virtually every Purkinje cell in the human cerebellum possesses numerous primary dendrites.
Further examinations in mice revealed that around half of their Purkinje cells also demonstrate this more intricate structure. Among these cells, 25% accept input from multiple climbing fibers, each connecting to different primary dendrite branches. Experiments monitoring cell activity in living mice also showed that primary branches can respond individually to various environmental stimuli.
“The longer you familiarize yourself with a particular cell model, the more you accept it,” stated Christian Hansel, Ph.D., Professor of Neurobiology at UChicago and senior author of the study, referring to the traditional belief that Purkinje cells have one main dendrite connected with one climbing fiber. “Cajal’s drawings have been around since the 1900s, so we certainly had ample time to notice, but only now with this quantitative analysis can we see that it’s almost universal that human cells have several fully-developed dendrites, and we can also observe its qualitative significance.”
Redefining a textbook concept
The cerebellum (from the Latin for ‘little brain’) is located at the base of the skull, just above the point where the spinal cord connects. Since French physician Jean Pierre Flourens first characterized the cerebellum’s function in 1824, scientists believed its only role was coordinating movement and muscular activity. However, advances in technology have revealed that the cerebellum also plays a significant part in processing information about the body’s internal and external environment, including sensations of proprioception and balance.
Cerebellar Purkinje cells function as large antennas, receiving thousands of signals containing a variety of information from the rest of the body. These signals are then integrated with a prediction-error signal, which indicates a discrepancy between the context and the brain’s expectation. This error signal is supplied by nerve fibers known as “climbing fibers,” which ascend from the brain stem and connect with their target Purkinje dendrite structures.
The prevailing understanding of these connections is that each Purkinje cell has a single primary dendrite that branches off from the cell body and connects with one climbing fiber, creating a singular computational unit. This one-to-one relationship belief between climbing fibers and Purkinje cells, a central principle in the field found in every neuroscience textbook, largely stems from studies on rodents, which predominantly exhibit the single dendrite configuration.
Mouse Purkinje cells. While 50% of mouse Purkinje cells have a single primary dendrite, the remaining half have multiple dendrites akin to human cells. Credit: Silas Busch, University of Chicago
Past studies examining these structures have typically focused on a small number of cells. Therefore, for this new research, Silas Busch, a Ph.D. student in Hansel’s lab and the paper’s first author, began by investigating thousands of cells from both human and mouse tissue. He utilized a specialized, antibody-based staining technique, known as immunohistochemistry, to selectively mark Purkinje cells in thin slices of cerebellum. He subsequently classified the structure of all observable cells and discovered that over 95% of human Purkinje cells contained multiple primary dendrites, whereas in mice this figure was roughly half.
Busch commented on the pervasiveness of the field’s established idea, noting that they’re even referred to as the ‘primary’ dendrite of a cell. “So, even the description of the structure of these cells is based on that mouse model that has one dendrite you can call a primary dendrite.”
Busch and Hansel were intrigued by this significant species difference in one of the most evolutionarily preserved brain areas shared across mammals and other vertebrates, which led them to question if there might be functional implications to possessing multiple primary dendrites rather than just one. Their first suspect was the climbing fiber, due to its significant one-to-one relationship and the intricate interweaving of the primary dendrite.
Through the use of sections of mouse cerebellum containing still living cells, Busch injected the cells with dye to visualize their branches and then stimulated the climbing fiber inputs. He noted that 25% of cells with multiple primary dendrites received multiple climbing fibers, challenging the textbook concept that each and every Purkinje cell only receives one climbing fiber input, while cells with a single primary dendrite did not.
Stimulating Whiskers and Observing Walking Mice
Boosted by the discovery that a substantial portion – though a minority – of Purkinje cells with multiple primary dendrites also receive input from multiple climbing fibers, Busch conducted a series of experiments in living mice to determine if it resulted in functional differences. He began by injecting a fluorescent calcium indicator dye into the cerebellum and implanting a small glass window for future observation of calcium flow into the Purkinje cell dendrites. By immobilizing the mouse’s head under a microscope while it ran on a treadmill, he was able to measure calcium flow that indicated when a climbing fiber was delivering a strong input to the cell. In cells with one primary dendrite, high-resolution images showed uniform activity signal across the structure; in cells with multiple primary dendrites, he detected activity on each side occurring at different times, indicating that one dendrite could be activated by its climbing fiber while the other dendrite in the same cell was not.
Next, Busch decided to see if he could distinguish individual climbing fiber activity using a more precise stimulus: the mouse’s whiskers. For this experiment, however, Busch had to sedate the mice. With the mice asleep, Busch inserted individual whiskers into a small glass tube and oscillated them back and forth. Here, he could also detect activity in specific dendritic branches of the Purkinje cells, suggesting that individual climbing fibers were relaying the input from individual whiskers to individual dendrites.
Lastly, for a more real-world scenario, Busch also tested awake mice with various stimuli, such as light flashes, sounds, or air puffs on the whisker pad. Once again, he saw differences across the Purkinje cells. In some, one branch might be particularly responsive to light but not sound, while another branch might be preferentially responsive to sound but not light.
“This occurred in a minority of cells since fewer have multiple branches in mice,
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Frequently Asked Questions (FAQs) about cerebellar connections
Q: What did the recent study reveal about cerebellar connections?
A: The recent study from the University of Chicago revealed that almost all human Purkinje cells in the cerebellum have multiple primary dendrites, contrary to the long-held belief of having a single main dendrite. This complexity in dendritic connections enables interactions with multiple climbing fibers originating from the brain stem.
Q: Why were early portrayals of Purkinje cells in the cerebellum significant?
A: Early portrayals by Santiago Ramón y Cajal depicted multiple large branches sprouting from Purkinje cells’ cell bodies, forming beautiful leaf-like patterns. However, despite these depictions, the consensus among neuroscientists was that Purkinje cells have only one primary dendrite connected to one climbing fiber.
Q: How did the recent study challenge the traditional understanding of cerebellar connections?
A: The recent study used advanced imaging techniques to examine thousands of cells from both human and mouse tissue. It revealed that the majority of human Purkinje cells have multiple primary dendrites, in contrast to the single dendrite configuration typically observed in mice.
Q: What did experiments on live mice reveal about the functional consequences of multiple primary dendrites?
A: Experiments on live mice showed that Purkinje cells with multiple primary dendrites could receive input from multiple climbing fibers. This indicated that different branches of the cells could be activated independently, responding to different stimuli from the environment.
Q: How might this new understanding of cerebellar connections have implications for diseases?
A: The new evidence of non-canonical connectivity in the cerebellum opens up possibilities for understanding diseases related to these structures. Previous studies have shown weaker Purkinje-climbing fiber connections in mouse models of cerebellar ataxia (a movement disorder) and stronger connections in genetic duplication and overexpression models of autism.
Q: What does the discovery of multiple primary dendrites suggest about the cerebellum’s role beyond motor coordination?
A: The presence of multiple primary dendrites in Purkinje cells indicates that the cerebellum’s role goes beyond just coordinating movement and muscular activity. It likely plays a significant part in processing information about the body’s internal and external environment, including sensations of proprioception and balance.
More about cerebellar connections
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“Scientists Discover Unexpected Complexity of Cerebellar Connections” – University of Chicago: Link
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“Climbing fiber multi-innervation of mouse Purkinje dendrites with arborization common to human” – Science Journal: Link
