| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2004: ¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 2003: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Research Abstract |
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.
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