| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
The in vitro transplacental perfusion method (IVTP: Suzue, 1990) made it possible for the first time to maintain live mammalian embryos with the uterus outside the maternal body. IVTP, therefore, appears to provide excellent opportunities to analyze development of mammalian embryos, because precise manipulation and continuous observation is possible. Taking the advantage of IVTP, in the present study, we attempted to establish a method for the manipulation and realtime observation of developing individual CNS neurons in live mammalian embryos. Mouse embryos at midgestation were maintained in vitro with IVTP and cells in forebrain and other CNS regions were labelled with Dil or DiO. Cells were observed with a fluorescence microscopy through the objective lens placed close to the surface of embryonic brain. The dyes brightly stained cells near the ventricles and radial fibers. By applying brief pulses of light intermittently, it became possible to observe migration of cells in embryonic brain for several hours. It was found that the populations of cells that show highly active migration in the embryonic brain were relatively small in the present conditions. Since this reduced migratory activities may be attributed to that some of the conditions of IVTP was not fully optimized for neuronal migration, we tried to improve the conditions of IVTP. After searches for conditions of nutrients, hormones and oxygen, we were able to make a great improvement for the living conditions of embryonic CNS neurons. These results show that there exist large variations for living conditions of embryos that can be altered independently by the difference in levels of various physiological factors. Therefore, these findings suggest that IVTP provide a great opportunity for studying various fields of mammalian embryonic development including the development of neurons, the factors that contribute normal development and abnormal development such as intrauterine growth retardation.
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