LTD and spatio-temporal dynamics of IP_3 within cerebellar Purkinje cells

平行纤维-普氏细胞突触的长期抑制（LTD）是小脑运动学习的基本电生理过程。虽然了解LTD的机制是一个需要解决的重要问题，但其分子机制的细节仍有待阐明。在本研究中，我们假设浦肯野细胞内肌醇1,4,5-三磷酸（IP_3）的胞内动力学调节了LTD的诱导，并解决了这一问题。我们使用GFP-PHD分析了突触前刺激如何影响浦肯野细胞的IP_3动力学，这是我们之前开发的用于可视化细胞内IP_3时空动力学的GFP-PHD。在双光子激光扫描显微镜下，用GFP-PHD转导小脑片制备中的浦肯野细胞。通过这种结构，我们可以看到浦肯耶细胞细树突中IP_3浓度的变化，我们首次成功地直接观察到平行纤维刺激时IP_3浓度的升高。谷氨酸受体有两种类型：离子型（iGluR）和代谢型（mGluR）谷氨酸受体。我们发现除了mGluR外，iGluR也参与了平行纤维输入引起的IP_3的产生。结合其他实验结果，我们得出结论，mGluR和iGluR分别通过g蛋白依赖机制和钙内流依赖机制协同作用IP_3的产生。这些研究结果大大促进了对IP_3在LTD调控中的时空作用的认识。我们还应该注意到，我们在研究过程中开发了几种新技术，包括RNAi文库构建方法和高效敲除IP_3受体的方法。这些技术将极大地促进对LTD分子机制的理解，以及相关领域的研究。

Long-term depression (LTD) at parallel fiber-Pukinje cell synapse is an elemental electrophysiological process underlying cerebellar motor learning. Although the understanding the mechanism of LTD is an important issue to be addressed, details of the molecular mechanism remain to be clarified. In this study, we hypothesized that the intracellular dynamics of inositol 1,4,5-trisphosphate (IP_3) within Purkinje cells regulates induction of LTD, and addressed the issue. We analyzed how presynaptic stimulations impact the IP_3 dynamics in Purkinje cells using GFP-PHD which we have previously developed for visualization of spatio-temporal dynamics of intracellular IP_3. Purkinje cells in cerebellar slice preparation transduced with GFP-PHD were imaged under the two-photon laser scanning microscope. With this configuration, allowing us to visualize changes in the concentration of IP_3 in the fine dendrites of Pukenje cells, we for the first time succeeded in direct observation of the raise in the concentration of IP_3 in response to parallel fiber stimulation. There are two types of glutamate receptors : ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. We discovered that not only mGluR but also iGluR participates in the production of IP_3 evoked by parallel fiber input. Taken together with the results of the additional experiments, we concluded that mGluR and iGluR, cooperatively work for IP_3 production through the G-protein and calcium influx dependent mechanisms, respectively. Theses research results thus greatly advances the understating of the spatio-temporal role of IP_3 in the regulation of LTD. We should also note that we have developed several new technologies including a method for RNAi library construction and a method for highly efficient knock down of IP_3 receptors in the course of this research. These technologies will greatly contribute to understanding of molecular mechanisms of LTD, as well as to the research in the related fields.