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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.

用突触前、后神经元的胞内荧光染色和突触前终末的免疫细胞化学染色，研究了分离培养的大鼠小脑浦肯野细胞上突触的空间分布。浦肯野电池是全电池电压钳制的，充满了德克萨斯红。然后，将其固定并用抗突触小泡的单抗进行FITC染色。染色结果显示，浦肯野细胞接受胞体和树突上的突触输入。在突触前小神经元和浦肯野细胞上同时进行全细胞记录，以确定突触传递的性质。同时，突触前神经元被荧光黄填充，浦肯野细胞被德克萨斯红填充。轴突肿胀被认为是兴奋性颗粒细胞的突触终末，仅分布在浦肯野细胞的树突上，而抑制性中间神经元的突触终末可能存在于树突和胞体上。用扫描电子显微镜观察突触。用免疫细胞化学方法检测mGluR1在浦肯野细胞上的分布。MGluR1在培养的浦肯野细胞的树突上呈点状分布，mGluR1分布在树突上多于胞体。这种分布模式与颗粒细胞兴奋性谷氨酸能突触的分布模式一致。因此，在一个简单的培养系统中，已经建立了该受体的细胞位置特异性突触形成和细胞位置特异性分布。