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

小脑浦肯野神经元发育早期分化的分子和生理学特征

• 批准号: 426359017
• 负责人: Friederike Stephani
• 金额: --
• 依托单位: Department of Neuroscience
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426359017?language=en
• 关键词: Molecular; physiological; characterization; differentiation; cerebellar

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.

小脑是后脑中的神经元结构，其主要涉及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，在很大程度上忽略了它的组织成不同的旁脑区室，特别是在有关的生理特性和功能。浦肯野细胞是抑制性GABA能神经元，其提供小脑的唯一输出，并且在所谓的分子层中配备有大量树突。浦肯野细胞可以分化成两种类型的zebrin-Ⅱ，它给出了一个特征性的条纹表达模式，在小脑划定旁组织，当染色。然而，目前还不清楚为什么存在两种不同类型的浦肯野细胞。最近首次显示，与不表达zebrin II（ZII+ PC）的浦肯野细胞（PC）相比，表达zebrin II（ZII+ PC）的浦肯野细胞（PC）之间的生理参数（例如放电率和规律性）不同。基于这些研究结果，我们假设ZII+和ZII-浦肯野细胞在其蛋白质组组成以及电生理特性方面存在显著差异。在这个提议中，我们的目标是表征培养的ZII+和ZII-PC，分离在胚胎第E18天，当小脑是相对不成熟的。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组成。基于发射率和zeolite II表达的差异，我们假设ZII+和ZII-PC之间的蛋白质组组成将更广泛地不同。第一个候选基因之一是TRPC 3，其在PC中高度表达，其中它有助于ZII-PC中的简单尖峰活性。因此，我们的目标是表征两种TRPC 3小鼠模型中的ZII+和ZII-PC，这两种TRPC 3小鼠模型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。利用siRNA敲除，我们将研究基因特异性敲除对ZII+和ZII- PCS的分化和行为的影响，这将使用不同的电生理参数以及免疫组织化学标记来测试。理解遗传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。这项研究将作为未来涉及浦肯野细胞分化的项目的基础，从而有助于理解小脑网络是如何形成的，并提供对不同疾病模型的更好理解。