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Neurite outgrowth in mature cerebral cortex investigated by a slice culture method.

通过切片培养方法研究成熟大脑皮层中的神经突生长。

基本信息

批准号：
07808090
负责人：
YAMADA Katsuya
金额：
$ 1.34万
依托单位：
Akita University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07808090/
关键词：
ADULT BRAIN SLICE CULTURE 熟成脳大脳皮質成熟神経細胞組織培養酸素分圧

项目摘要

To make culture of mature cerebral cortex has been thought to be difficult. Previously, I showed that a part of neurons in 3-4 week-old rat visual cortex can survive for 1 week by making very thin (150 micron) slice. In that case, however, I had only observed neurite outgrowth from and within the slice using fluorescent dyes. It was difficult to observe field potentials by stimulating underlying white matter in a very thin slice. In the present study, I made 400 microns-thick cortical slices. This enabled us to estimate slice viability from electrophysiological aspect.Population field responses in mature cortical slices disappeared within 4 hours when they were put on a conventional interface-type culture dish in a commerciallyavailable CO2 incubator. Most of the neurons soon showed apparent shrinkage. In contrast, slices which were continuously perfused by culture medium (1.2ml/min) showed field responses over 18 hours. With our previous culture preparing method, considerablly long period was needed for atmosphere in a culture dish to reach equibrium in gas composition, pH,humidity, temperature after they are exposed in room air at the instance of transferring tissues. This may be fatal for adult cortical tissues. Now I am constructing a new system which enables us to conduct a long time recording in a sterilized condition.

成熟大脑皮层的培养一直被认为是困难的。在此之前，我已经证明，在3-4周龄大鼠的视觉皮层中，通过制作非常薄（150微米）的薄片，部分神经元可以存活一周。然而，在这种情况下，我只使用荧光染料观察了切片上和切片内的神经突生长。在非常薄的薄片上刺激下层白质很难观察到场电位。在目前的研究中，我制作了400微米厚的皮层切片。这使我们能够从电生理方面估计切片的生存能力。将成熟皮层切片置于常规界面型培养皿和商用CO2培养箱中，4小时内种群场反应消失。大多数神经元很快就表现出明显的萎缩。以1.2ml/min的培养基连续灌注18小时后，切片呈现出田间反应。用我们以前的培养制备方法，在转移组织时，培养皿中的气体成分、pH值、湿度、温度暴露在室内空气中，需要相当长的时间才能达到平衡。这对成人皮质组织可能是致命的。现在我正在构建一个新的系统，使我们能够在无菌的条件下进行长时间的记录。