Role of IP_3 in LTD in cerebellar purkinje cells

本研究的目的是阐明肌醇la5 -三磷酸（IP_3）在诱导长时间抑郁中的作用，特别是IP_3信号的时空方面。为了实现这一目标，我们开发了新的技术，使我们能够可视化小脑浦肯野神经元中IP_3的细胞内动力学。我们使用GFP- phd作为IP_3成像探针，GFP- phd是由GFP和磷脂酶C δ1的pleckstrin同源结构域组成的融合蛋白。我们首次证明了IP_3在GFP-PHD介导的浦肯野神经元中可见。此外，我们将含有编码GFP-PHD的Sindbis病毒载体直接注射到小鼠脑内，成功地将GFP-PHD导入到小脑切片浦肯野神经元中。小脑切片解剖后发现GFP-PHD具有较高的转导效率。利用这些新技术，我们发现AMPA受体激活可诱导培养的浦肯野神经元细胞内IP_3的增加。AMPA受体介导的IP_3的产生并不是单纯的副现象，因为在小脑片制备过程中，AMPA受体通过攀爬纤维刺激激活也引起了浦肯野神经元IP_3的产生。因此，我们的发现对于理解LTD诱导机制具有重要意义。本研究的目的是阐明肌醇la5 -三磷酸（IP_3）在诱导长时间抑郁中的作用，特别是IP_3信号的时空方面。为了实现这一目标，我们开发了新的技术，使我们能够可视化小脑浦肯野神经元中IP_3的细胞内动力学。我们使用GFP- phd作为IP_3成像探针，GFP- phd是由GFP和磷脂酶C δ1的pleckstrin同源结构域组成的融合蛋白。我们首次证明了IP_3在GFP-PHD介导的浦肯野神经元中可见。此外，我们将含有编码GFP-PHD的Sindbis病毒载体直接注射到小鼠脑内，成功地将GFP-PHD导入到小脑切片浦肯野神经元中。小脑切片解剖后发现GFP-PHD具有较高的转导效率。利用这些新技术，我们发现AMPA受体激活诱导浦肯野神经元细胞内IP_3的积累。AMPA受体介导的IP_3的产生并不是单纯的副现象，因为在小脑片制备过程中，AMPA受体通过攀爬纤维刺激激活也引起了浦肯野神经元IP_3的产生。因此，我们的发现对于理解LTD诱导机制具有重要意义。少

The aim of the present research project was to clarify the role of inositol lA5-trisphosphate (IP_3) in the induction oflong tem depression, especially focusing on the spatio-temporal aspects of IP_3 signaling. To achieve this end, we have developed novel technologies that enable us to visualize the intracellular dynamics of IP_3 in cerebellar Purkinje neurons. As an IP_3 imaging probe, we employed GFP-PHD which is a fusion protein consisting of GFP and pleckstrin homology domain of phospholipase C δ1. We first proved that IP_3 can be visualized in cultured Purkinje neurons which is transduced with GFP-PHD. Furthermore, by directly injecting Sindbis virus vector containing cDNA encoding GFP-PHD into the mouse brain, we successfully introduced GFP-PHD into Purkinje neurons in cerebellar slice. The transduction efficiency of GFP-PHD was found to be high after dissection of cerebellar slices. Using these novel techniques, we found that AMPA receptor activation induces intracellular accumu … More lation of IP_3 in cultured Purkinje neuron. AMPA receptor-mediated IP_3 production was not a mere epiphenomenon, because AMPA receptor activation through climbing fiber stimulation also elicited IP_3 production in Purkinje neuron in cerebellar slice preparation. Our findings are thus important in an understanding of the mechanism of LTD induction.The aim of the present research project was to clarify the role of inositol lA5-trisphosphate (IP_3) in the induction oflong tem depression, especially focusing on the spatio-temporal aspects of IP_3 signaling. To achieve this end, we have developed novel technologies that enable us to visualize the intracellular dynamics of IP_3 in cerebellar Purkinje neurons. As an IP_3 imaging probe, we employed GFP-PHD which is a fusion protein consisting of GFP and pleckstrin homology domain of phospholipase C δ1. We first proved that IP_3 can be visualized in cultured Purkinje neurons which is transduced with GFP-PHD. Furthermore, by directly injecting Sindbis virus vector containing cDNA encoding GFP-PHD into the mouse brain, we successfully introduced GFP-PHD into Purkinje neurons in cerebellar slice. The transduction efficiency of GFP-PHD was found to be high after dissection of cerebellar slices. Using these novel techniques, we found that AMPA receptor activation induces intracellular accumulation of IP_3 in cultured Purkinje neuron. AMPA receptor-mediated IP_3 production was not a mere epiphenomenon, because AMPA receptor activation through climbing fiber stimulation also elicited IP_3 production in Purkinje neuron in cerebellar slice preparation. Our findings are thus important in an understanding of the mechanism of LTD induction. Less