Cerebellar Neurogensis

描述(由申请人提供)：这项建议将测试谷氨酸能信号协调颗粒细胞发育的早期阶段的假说，这是正常小脑回路形成所必需的。谷氨酸跨门具有保守的信号传递功能，通过不同的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，我们对这些功能的了解落后于已知的谷氨酸受体亚型的多样性。以往的和初步的数据表明，颗粒细胞前体(GCP)在退出细胞周期并延伸轴突时，在外生发层(EGL)瞬时表达功能性的含GluK2的海人酸受体(KAR)和I组代谢性谷氨酸受体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会改变GCP的发育，如果是的话，又是如何改变的。在这项提案中，目标1和目标2将分别研究GluK2和mGlu5R在GCP增殖和轴突延伸中的作用。目的3探讨Bergmann胶质细胞是否通过mGlu5R释放谷氨酸，控制GCP细胞周期的退出。我们将在急性切片和体内方法(药物、电穿孔RNA干扰和遗传操作)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)，包括野生型和转基因小鼠的mGlu5Rfl/fl(f1，Floxed)和GluK2基因敲除(KO)。

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).