Study of the calcium dynamics and molecular mechanisms in the dendrite of cerebellar Purkinje cell

我们研究了神经元树突中IP 3受体（IP 3R）的动态和功能。由于已知IP 3R在突触后的突触可塑性中起关键作用，因此IP 3R进出树突棘的运输变化将影响突触可塑性的潜在机制。通过基因枪基因转染技术和双光子显微镜技术，我们比较了IP 3R和另一种ER膜蛋白SERCA在小脑浦肯野细胞棘中的侧向扩散效率。我们发现，IP 3R的扩散效率是显着低于SERCA的，这表明了一种机制，即丰富的IP 3R蛋白在棘，由于其高亲和力的肌动蛋白细胞骨架已显示在海马锥体细胞的树突由我们以前。在另一个研究IP 3R在小脑皮质的功能的方法中，我们发现IP 3R在浦肯野细胞树突的形成中起作用。出乎意料的是，在小脑颗粒细胞中表达的IP 3R是关键的参与者，而不是在浦肯野细胞中高表达的IP 3R。这一结果显示了IP 3R在小脑皮质中的另一个重要作用。我们还成功地通过用荧光蛋白工程化IP 3R的IP 3结合部分来创建IP 3指示剂，其清楚地显示了COS-7细胞系中钙振荡期间的IP 3振荡。这种新的指示剂将是一种有效的工具，研究IP 3和IP 3R在突触可塑性中起重要作用的神经元树突中的IP 3动力学。

We investigated dynamics and functions of IP3 receptor (IP3R) in the neuronal dendrite. Since IP3R is known to play a pivotal role in synaptic plasticity in the postsynapse, changes in the trafficking of IP3R into/out of dendritic spines would affect the mechanism underlying synaptic plasticity. We compared lateral diffusion efficacy of IP3R with another ER membrane protein, SERCA, in spines of cerebellar Purkinje cells by expressing genes of the ER proteins fused with fluorescent proteins by means of gene gun gene delivery technique and two photon microsoopy. We found that the diffusion efficacy of IP3R was significantly lower than that of SERCA, suggesting a mechanism which enriches IP3R proteins in spines due to their high affinity to the actin cytoskeleton as has been shown in the dendrites of hippocampal pyramidal cells by us previously. In another approach to the IP3R function in the cerebellar cortex, we found that IP3R plays a role in formation of the dendrite of Purkinje cell. It was unexpected that IP3R expressed in the cerebellar granule cells was the key player instead of the highly expressed IP3Rs in the Purkinje. This result shows another important role of IP3R in the cerebellar cortex. We also succeeded to create an IP3-indicator by engineering the IP3-binding part of IP3R with fluorescent proteins, which clearly showed IP3 oscillation during calcium oscillation in the COS-7 cell line. This novel indicator will be a potent tool investigate IP3 dynamics in neuronal dendrites where IP3 and IP3R play important roles in synaptic plasticity.