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.
