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Cerebellar modules and the Ontogeny of Sensorimotor Integration

Periodic Reporting for period 4 - COSI (Cerebellar modules and the Ontogeny of Sensorimotor Integration)

Reporting period: 2020-12-01 to 2021-11-30

The perfect execution of a voluntary movement requires the integration of the current position of your body, information from your environment and the desired outcome. To assure that this motor output becomes and remains appropriate, the brain needs to learn from the result of previous outputs. The little brain, or cerebellum, plays an important role in this process called sensorimotor integration, yet -despite decades of studies- there is no generally excepted theory for how the cerebellum functions.

When the function of the cerebellum is disrupted, for instance due to tumor or stroke, patients have enormous difficulties in speech, walking, maintaining balance, making directed movement, etc. These problem also occur as the result of mutations in certain genes, leading to e.g. spino-cerebellar ataxias (SCAs) and more recently the cerebellum has also been linked to neurodevelopmental disorders, for instance autism spectrum disorder (ASD). In order to provide to basis for understanding how these disorder result in their specific, sometimes very different problems, we study the functioning of the cerebellum.

We and others recently found that, although the cerebellum is a single structure, it actually consists of at least two distinct circuits that differ in many aspects. First, the main cell in the circuit, the Purkinje cell, can be use that separate the circuits by looking at their gene expression. Second, when comparing subpopulations divided based on gene expression, differences in the activity of the Purkinje cells was observed. Moreover, in several mouse models for known disorders and naturally occurring variation one of the two populations is specifically affected. Although all this knowledge together supports the view of the cerebellum consisting of two separate circuits, a deeper understanding of the differences between the two circuits and how they develop is needed. The goal of the current project is to determine how the cerebellum functions and dysfunctions through a deeper analysis of the differences between cerebellar neurons, with a particular focus on the development of the brain. Our hypothesis is that these differences will explain existing controversies, and unify contradicting results into one central theory on cerebellar function.

To this end, we are working on three key objectives. First, we will look at the development of the connections and information flow towards and within the cerebellum, and determine if this differs between the two subpopulations (key objective A). Next, we will compare the two subpopulations in more detail, looking at the genes they express and how these determine their level of activity (key objective B). Finally, we will determine how the differences in connections and gene expression affect how the two subgroups control behavior (key objective C). This will be evaluated with more standard task testing motor coordination and learning, but also by testing more social and cognitive functions, as these functions in recent years have also been linked to the cerebellum.

By combining the information from these three key objectives, we aim to determine how the two subgroups develop and contribute to the different functions that the cerebellum is involved in. The ultimate goals is to produce a unifying theory for cerebellar functioning based on the development of the two subgroups. This knowledge is fundamental for the diagnosis and treatment of cerebellum-related neurodegenerative disorders, including SCAs, but also to understand the contribution of the cerebellum to the neurodevelopmental disorder, such as ASD.
With respect to the three key objectives stated above the following results have been obtained:

Key objective 1:
We have addressed the goal of studying the inputs to the cerebellar cortex and cerebellar nuclei first by analyzing the development of projections from the cortex, via Purkinje cell (PC) axons, to the cerebellar nuclei. In this analysis we differentiated two populations of PCs, based on the expression of the protein ZebrinII (or Aldoc). We found that as early P10 a difference in complexity of the axonal arborization can be observed, with a larger, more complex arborization of the ZebrinII-positive (Z+) PCs (Beekhof et al., 2021). In addition, we have and are currently still examining the temporal and spatial profile of connectivity of other mossy fiber sources. We have identified specific markers that can be used to label subpopulations of mossy fibers. One is particularly interesting, labeling a subpopulation that specifically targets a single module of Purkinje cells, the 9+ band.

Key objective 2:
We have identified and investigated in detail the contributions of various proteins, both in the normal developing system as well as in disease. We have determined the development of the intrinsic activity of PCs, the activity in vivo when all inputs are present, together with the development of the morphology of PCs. We found that clear differences between Z+ and Z- at the adult stage are already present early in development, and that the timelines of development are distinctly different. Interestingly, more rapid development in Z+ PCs correlated with, relative to adult mice, better learning skills at 3 weeks of age, while Z- behavior did not show this learning enhancement (Beekhof et al., 2021, Elife). We also tested the role of TRPC3 and SK2 and identified their roles in Purkinje cell activity, predominantly in Z- Purkinje cells, and their contributions to behavior (Wu et al, 2019 and Grasselli et al., 2020). To assess if and how differentiation of PC subpopulations are relevant in cerebellar disease we studied disease progression in spinocerebellar ataxia type1 (SCA1, White et al., 2021, Brain Pathology) and tested the role of sphingosine kinase 1 (Sphk1) in disease progression. Deletion of Sphk1, a key enzyme in the equilibrium of ceramide, sphingosine and S1P, in the SCA1 mouse model resulted in a rescue of disease progression (Blot et al., 2021).

Key objective 3:
We have studied the development of behavior and the contributions of various genes in several key experiments. In a first step towards conversion of Purkinje cell activity to behavioral relevance, we recorded the activity of their downstream targets, cerebellar nuclear neurons. Receiving only input from one type of Purkinje cell, Z+ or Z-, we tested of higher levels of input from Z- PCs reduced activity in cerebelar nuclei neurons. Remarkably, we found the opposite, resulting in the conclusion that all three elements of the loop: inferior olive neurons, PCs and cerebellar nuclei neurons, which are linked to Z- PCs have a higher firing rate than those linked to Z+ PCs (Beekhof et al., 2021). Moreover, we are currently completing two manuscripts on the differential contribution of distinct subpopulations of PCs to behavior. To this end, we have identified new markers, for optimal differentiating in areas where ZebrinII is less specific and we have found specific Cre-lines that allow for targeted manipulation of subpopulations in small zones (microzones). Moreover, we have developed a novel behavioral task for rapid, cerebellar-dependent learning of full-body movement. This task will allow us to test the PC response related to the unconditioned and conditioned response across multiple microzones.

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