|Budget Amount *help
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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.