The development of neural circuits, as exploited by whole-embryo culture

全胚胎培养所利用的神经回路的发育

• 批准号: 12670028
• 负责人: YAMAKADO Makoto
• 金额: $ 1.6万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670028/
• 关键词: whole-embryo culture; facial vibrissae; trigeminal ganglion; DiI; neural circuits; neural development; パイオニア線維; ヒゲ原基; 足場; 標的認知; 誘導

In order to examine the development of vibrissal connections in culture, we first tested the validity of whole-embryo culture technique as a tool for the experiment under close and continuous observation in mouse embryos. Since vibrissal rudiments begin to develop at E11.5, embryos at E11.5 and E12.5 were explanted to culture for 4h, 6h 24h or 48h, respectively. The growth of embryos in culture was assessed by the heart rate, crown-rump length and total number of somites. DiI tracing method in whole-mount preparations fixed with 4% paraformaldehyde evaluated the neural connection with vibrissal rudiments at the end of the culture period. In cases of 24h in culture, it was realized that the development of vibrissal rudiments, distribution of innervating nerve fibers, and typical ending structures at the rudiments could be maintained satisfactorily to define. In contrast, in cases of 48h in culture, a certain retraction was found in the fasciculation of nerve fibers innervating the rudiments. These results indicate that whole-embryo culture is the most available tool to exploit the mechanisms of neural connections for targeted structures in embryos, although confined during the period of 24h. At present, it is not succeeded to develop the technique for the image analysis in culture condition. To establish the system apparatus to analyze neural mechanisms at molecular level using whole-embryo culture is the next step for our study project.

为了检测胚胎培养中振动连接的发育，我们首先在小鼠胚胎中检验了全胚胎培养技术作为实验工具在近距离和连续观察下的有效性。E11.5和E12.5的胚胎分别在E11.5、E12.5和E11.5开始发育，分别培养4h、6h、24h和48h。通过心率、冠臀长度和体节总数来评价胚胎在培养过程中的生长情况。4%多聚甲醛固定的整装制剂的DiI示踪法在培养期末评估神经与振动雏形的联系。在培养24小时的情况下，可以很好地维持振动雏形的发育、支配神经纤维的分布和雏形上典型的末端结构。相反，在培养48h的情况下，发现支配原基的神经纤维束有一定的回缩。这些结果表明，全胚胎培养是探索胚胎靶向结构神经连接机制的最有效工具，尽管仅限于24小时。目前，文化条件下的图像分析技术还没有得到成功的发展。建立利用全胚胎培养在分子水平上分析神经机制的系统装置是我们下一步的研究项目。