Cerebellar Neurogensis

描述（由申请人提供）：该提案将测试谷氨酸能信号协调颗粒细胞发育的早期阶段的假设，这是小脑回路形成所必需的。谷氨酸具有保守的跨门信号功能，通过多种受体控制多种细胞内信号通路。谷氨酸受体在大脑中的表达受到严格的发育调控，这表明每种受体亚型在细胞发育的不同阶段具有特定的功能。事实上，新出现的证据表明谷氨酸受体与大脑发育有关。然而，我们对这种功能的理解落后于已知的谷氨酸受体亚型的多样性。先前和初步的数据表明，颗粒细胞前体（GCPs）在离开细胞周期并延伸轴突时，会在外生发层（EGL）短暂表达含有gluk2的kainate受体（KAR）和I组代谢型谷氨酸受体5 （mGlu5R）。然而，目前尚不清楚GluK2受体和mGlu5R功能的获得或丧失是否以及如何改变GCP的发展。在本提案中，Aim 1和Aim 2将分别检测GluK2和mGlu5R对GCP增殖和轴突延伸的功能。Aim 3将探讨Bergmann胶质细胞（一种特殊的星形胶质细胞，通过EGL扩展过程）是否通过mGlu5R释放谷氨酸控制GCP细胞周期退出。我们将在野生型和转基因小鼠（包括mGlu5Rfl/fl （fl, floxed）和GluK2-敲除（KO））的急性切片和体内方法（药物、通过电穿孔进行RNA干扰和基因操作）中结合使用延时共聚焦显微镜（用于钙成像、迁移和轴突延伸）。

DESCRIPTION (provided by applicant): This proposal will test the hypothesis that glutamatergic signaling orchestrates early stages of granule cell development necessary for proper cerebellar circuit formation. Glutamate, which has a conserved signaling function across phyla, controls multiple intracellular signaling pathways through diverse receptors. Glutamate receptor expression in the brain is tightly developmentally regulated, suggesting specific functions of each receptor subtype at different stages of cell development. Indeed, emerging evidence implicates glutamate receptors in brain development. However, our understanding of such functions lags behind the known diversity of glutamate receptor subtypes. Previous and preliminary data suggest that granule cell precursors (GCPs) transiently express functional GluK2-containing kainate receptors (KAR) and group I metabotropic glutamate receptor 5 (mGlu5R) in the external germinal layer (EGL) when they exit the cell cycle and extend axons. However, it is not known whether, and if so how, gain or loss of function in GluK2 receptor and mGlu5R alters GCP development. In this proposal, Aim 1 and Aim 2 will examine the function of GluK2 and mGlu5R on GCP proliferation and axon extension, respectively. Aim 3 will explore whether Bergmann glial cells, a specialized astrocytes extending processes through the EGL, release glutamate controlling GCP cell-cycle exit through mGlu5R. We will use a combination of time- lapse confocal microscopy (for calcium imaging, migration, and axon extension) in acute slices and in vivo approaches (drugs, RNA interference via electroporation, and genetic manipulation) in wild-type and transgenic mice, including mGlu5Rfl/fl (fl, floxed) and GluK2- knockout (KO).