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)在长时程增强抑制诱导中的作用，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。我们用绿色荧光蛋白与磷脂酶Cδ1的Pleckstrin同源结构域的融合蛋白GFP-PhD作为IP_3的成像探针。我们首次证明了IP_3可以在用绿色荧光蛋白-PhD转导的培养的浦肯野神经元中可见。此外，将含有GFP-PhD编码基因的Sindbis病毒载体直接注射到小鼠脑内，成功地将GFP-PhD导入小脑片上的浦肯野神经元。切取小脑片后，GFP-PHD的转导效率较高。利用这些新技术，我们发现ampa受体的激活诱导了细胞内的蓄积…体外培养的浦肯野神经元IP_3含量增加。AMPA受体介导的IP_3的产生并不是一种单纯的现象，因为通过刺激攀升纤维激活AMPA受体也能在小脑切片制备的浦肯野神经元中诱导IP_3的产生。因此，我们的发现对于理解LTD的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)在长时相抑郁诱导中的作用，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。我们用绿色荧光蛋白与磷脂酶Cδ1的Pleckstrin同源结构域的融合蛋白GFP-PhD作为IP_3的成像探针。我们首次证明了IP_3可以在用绿色荧光蛋白-PhD转导的培养的浦肯野神经元中可见。此外，将含有GFP-PhD编码基因的Sindbis病毒载体直接注射到小鼠脑内，成功地将GFP-PhD导入小脑片上的浦肯野神经元。切取小脑片后，GFP-PHD的转导效率较高。利用这些新技术，我们发现AMPA受体激活诱导培养的浦肯野神经元细胞内IP_3积聚。AMPA受体介导的IP_3的产生并不是一种单纯的现象，因为通过刺激攀升纤维激活AMPA受体也能在小脑切片制备的浦肯野神经元中诱导IP_3的产生。因此，我们的发现对于理解LTD诱导的机制具有重要意义。较少

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