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

为了研究培养中触须连接的发育，我们首先在小鼠胚胎中测试了全胚胎培养技术作为实验工具的有效性，并进行了密切和连续的观察。由于胚胎发育开始的时间为E11.5，因此将E11.5和E12.5的胚胎分别培养4h、6h、24h和48h。通过心率、顶臀长和体节总数来评估胚胎在培养中的生长。DiI示踪法在4%多聚甲醛固定的整装制剂中评价了培养期结束时与触须雏形的神经连接。在培养24小时的情况下，发现触须雏形的发育、支配神经纤维的分布以及雏形处典型的末端结构都能令人满意地保持以确定。相反，在培养48小时的情况下，发现支配雏形的神经纤维的成束有一定的收缩。这些结果表明，全胚胎培养是最有效的工具，利用胚胎中的目标结构的神经连接的机制，虽然限制在24小时内。目前，在培养条件下进行图像分析的技术还没有发展成功。本研究的下一步工作是建立一套利用全胚胎培养技术在分子水平上分析神经机制的系统装置。

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.