Molecular and physiological characterization of the differentiation in cerebellar Purkinje neurons early in development

• Applicant: Friederike Stephani
• Subject Area: Cognitive, Systems and Behavioural Neurobiology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 426359017

The cerebellum is a neuronal structure in the hindbrain that is mainly involved in the coordination and integration of motor and cognitive processes. Cellular heterogeneity is essential to ensure that specific types of neurons differentiate in order to fulfill certain functions and form dedicated neuronal circuits. Over the past years the cerebellum has been considered to be a homogeneous unit, largely neglecting its organization into distinct parasagittal compartments, particularly in relation to physiological properties and function. Purkinje cells are inhibitory GABAergic neurons that provide the sole output of the cerebellum and that are equipped with massive dendrites in the so-called molecular layer. Purkinje cells can be differentiated into two types by zebrin-II, which gives a characteristic striped expression pattern in the cerebellum demarcating the parasagittal organization, when stained for. However it is unclear why two distinct types of Purkinje cells exist. Recently it was shown for the first time that physiological parameters, such as e.g. firing rate and regularity, differed between Purkinje cells (PCs) that express zebrinII (ZII+ PCs) compared to those that do not express it (ZII- PCs). Based on these findings, we hypothesize that ZII+ and ZII- Purkinje cells differ substantially in their proteomic composition as well as in their electrophysiological properties. In this proposal we aim to characterize cultured ZII+ and ZII- PCs, isolated at embryonic day E18, when the cerebellum is relatively immature. To do so, we will use whole-cell patch-clamp recordings and single-cell RNA-sequencing to study the proteomic composition. Based on the difference in firing rate and the expression of zebrin II, we hypothesize that the proteomic composition will more extensively differ between ZII+ and ZII- PCs. One of the first candidate genes is TRPC3, which is highly expressed in PCs, where it contributes to the simple spike activity in ZII- PCs. We therefore aim to characterize the ZII+ and ZII- PCs in two TRPC3 mouse models, representing a loss-of function and a gain-of-function mutation mouse model, with respect to electrophysiological properties and immunohistochemical marker. Using siRNA knockdown, we will study the influence of gene specific knockdown on the differentiation and behavior of ZII+ and ZII- PCS, which will be tested using different electrophysiological parameter as well as immunohistochemical labeling. It is essential to understand how the genetically driven differentiation results in the cellular heterogeneity that underlies different Purkinje cell activity. The study will serve as a basis for future projects involving Purkinje cell differentiation and thereby contribute to the understanding of how cerebellar networks are formed and in addition provide a better understanding of different disease models.

• DFG Programme: Research Fellowships
• International Connection: Netherlands
• Host: Dr. Martijn Schonewille