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

小脑是后脑的一个神经元结构，主要参与运动和认知过程的协调和整合。细胞异质性对于确保特定类型的神经元分化以实现某些功能并形成专用的神经元回路至关重要。在过去的几年里，小脑一直被认为是一个同质的单位，在很大程度上忽视了它的组织成不同的旁矢状隔室，特别是在生理特性和功能方面。浦肯野细胞是一种抑制性gaba能神经元，为小脑提供唯一的输出，在所谓的分子层上配备了大量的树突。斑马素- ii可将浦肯野细胞分化为两种类型，斑马素- ii在小脑中具有典型的条纹表达模式，用于划分副矢状面组织。然而，目前尚不清楚为什么存在两种不同类型的浦肯野细胞。最近首次发现，表达zebrinII （ZII+ PCs）的浦肯野细胞（PCs）与不表达zebrinII （ZII- PCs）的浦肯野细胞（PCs）之间的生理参数（如放电速率和规律性）存在差异。基于这些发现，我们假设ZII+和ZII-浦肯野细胞在蛋白质组学组成和电生理特性上存在很大差异。在本研究中，我们的目标是表征在胚胎期E18分离的培养的ZII+和ZII- PCs，当时小脑相对不成熟。为此，我们将使用全细胞膜片钳记录和单细胞rna测序来研究蛋白质组学组成。基于发射速率和zebrin II表达的差异，我们推测ZII+和ZII- PCs之间的蛋白质组学组成差异更大。第一批候选基因之一是TRPC3，它在pc中高度表达，在ZII- pc中参与简单的spike活性。因此，我们的目标是在两种TRPC3小鼠模型中表征ZII+和ZII- pc，代表功能丧失和功能获得突变小鼠模型，关于电生理特性和免疫组织化学标记。通过siRNA敲低，我们将研究基因特异性敲低对ZII+和ZII- PCS分化和行为的影响，我们将使用不同的电生理参数和免疫组织化学标记进行测试。了解基因驱动的分化如何导致不同浦肯野细胞活性的细胞异质性是至关重要的。这项研究将为涉及浦肯野细胞分化的未来项目奠定基础，从而有助于了解小脑网络是如何形成的，此外还有助于更好地了解不同的疾病模型。

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.