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

平行纤维-Pukinje细胞突触长时程抑制（LTD）是小脑运动学习的基本电生理过程。虽然对LTD的机制的理解是一个重要的问题，但其分子机制的细节仍有待澄清。在这项研究中，我们假设在浦肯野细胞内的肌醇1，4，5-三磷酸（IP_3）的细胞内动力学调节LTD的诱导，并解决了这个问题。我们使用我们先前开发的用于可视化胞内IP_3时空动力学的GFP-PHD来分析突触前刺激如何影响浦肯野细胞IP_3动力学。在双光子激光扫描显微镜下对转染GFP-PHD的小脑切片制备物中的浦肯野细胞进行成像。通过这种配置，我们可以观察Pukenje细胞细树枝状突起中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后IP_3浓度的升高。有两种类型的谷氨酸受体：离子型（iGluR）和代谢型（mGluR）谷氨酸受体。我们发现不仅mGluR参与了平行纤维输入诱发的IP_3的产生，iGluR也参与了这一过程。结合其他实验结果，我们认为mGluR和iGluR分别通过G蛋白和钙离子内流依赖的机制协同作用于IP_3的产生。这些研究结果大大推进了对IP_3在LTD调控中时空作用的理解。我们还应该注意到，在本研究过程中，我们开发了几种新技术，包括RNAi文库构建方法和高效敲除IP_3受体的方法。这些技术将有助于深入了解LTD的分子机制，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.