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Cell-site specific synapse formation in culture

培养物中细胞位点特异性突触的形成

基本信息

批准号：
04454134
负责人：
HIRANO Tomoo
金额：
$ 4.74万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454134/
关键词：
Synapse formation Cerebellum Culture Purkinje cell Granule cell Basket cell Stellate cell Fluorescent staining プルキン工細胞

项目摘要

Spatial distribution of synapses on Purkinje cells formed in a dissociated cell culture of rat cerebellum, was studied by intracellular fluorescent stainings of pre- and postsynaptic neurons and by immunocytochemical staining of presynaptic terminals. A Purkinje cell was whole-cell voltage-clamped and filled with Texas red. Then, it was fixed and stained with an anti-synaptic vesicle monoclonal antibody using FITC.This staining revealed that a Purkinje cell receives synaptic inputs on both a soma and dendrites. Simultaneous whole-cell recordings were performed on a presynaptic small neuron and a Purkinje cell, and the property of synaptic transmission was determined. At the same time, the presynaptic neuron was filled with Lucifer yellow and the Purkinje cell was filled with Texas red. Axonal swellings presumed to be synaptic terminals of an excitatory granule cell, were exclusively localized along dendrites of a Purkinje cell, and presumed synaptic terminals of an inhibitory interneuron were found on both dendrites and a soma. Synapses were also observed with a scanning electron microscope. Antibodies were raised against one subtype of glutamate receptors, mGluR1, and distribution pattern of mGluR1 on a Purkinje cell was examined with immunocytochemical staining. Punctate labelings of mGluR1 were observed along dendrites of cultured Purkinje cells.mGluR1 were distributed more on dendrites than on a cell body. This distribution pattern of mGluR1 is consistent with that of excitatory glutamatergic synapses from granule cells. Thus, cell-site specific synapse formations and also cell-site specific distribution of the receptor have been established in a simple culture system.

用细胞内荧光染色法和免疫细胞化学染色法研究了大鼠小脑浦肯野细胞突触的空间分布。浦肯野细胞是全细胞电压钳位的，并充满德克萨斯红。固定后，用抗突触囊泡单克隆抗体进行FITC染色，结果显示浦肯野细胞接受索马和树突的突触输入。同时进行全细胞记录突触前小神经元和浦肯野细胞，并确定突触传递的属性。同时，突触前神经元充满了荧光黄，浦肯野细胞充满了德克萨斯红。轴突soup推测是兴奋性颗粒细胞的突触末梢，仅定位沿着树突的浦肯野细胞，和推测的突触末梢的抑制性中间神经元上发现的树突和索马。用扫描电子显微镜观察突触。针对谷氨酸受体的一种亚型，mGluR1的抗体，并在浦肯野细胞上的mGluR1的分布模式进行了研究与免疫细胞化学染色。mGluR 1在培养的浦肯野细胞树突上呈沿着点状分布，在树突上的分布多于在胞体上的分布。mGluR1的这种分布模式与来自颗粒细胞的兴奋性突触的分布模式一致。因此，细胞位点特异性突触形成和受体的细胞位点特异性分布已经在简单的培养系统中建立。