Analysis of early infection with poliovirus in motor neurons.

我们利用双室系统成功地培养了大鼠和小鼠的原代运动神经元。它使我们能够在不同的培养液中培养突触侧和细胞体一侧。为了改善培养条件，我们对培养基条件进行了改进。加入小鼠骨骼肌提取物后，轴突延长得到改善。我们在双室培养人脊髓灰质炎病毒(PV)受体(HPVR)转基因(TG)小鼠的运动神经元，并在突触侧或细胞体侧加入编码GFP的PV缺陷干扰颗粒。当病毒从细胞体一侧加入时，加入后6h可观察到绿色荧光蛋白的表达。另一方面，当病毒从突触侧加入时，直到加入后24小时才观察到GFP的表达，而在加入后48小时才观察到表达。这些结果表明，来自细胞体一侧的感染与来自突触一侧的感染的进展不同。接下来，我们试图优化活荧光细胞的四维成像系统。为了在显微镜下长期培养，我们在封闭系统中将培养基换成了不含二氧化碳的培养基。这使得细胞能够在显微镜下存活更长时间。在激光共聚焦显微镜下，我们尝试了不同的激光强度、针孔大小、Z切片数等条件进行长期观察。但是，只要使用激光共聚焦显微镜，就不能优化长期观察的条件。

We were able to culture primary motor neurons from rat or mouse using dual chamber system. It enabled us to culture the synapse side and cell body side in the different medium. To improve the culture condition, we improved the condition of the medium. The axonal elongation has improved by adding the extract of skeletal muscles from mouse. We cultured motor neurons derived from human poliovirus (PV) receptor (hPVR) - transgenic (Tg) mouse on the dual chamber and added PV defective interfering particles coding GFP from the synapse side or the cell body side. When the virus was added from the cell body side, expression of GFP was observed at six hours after the addition. On the other hand, when the virus was added from the synapse side, expression of GFP had not been observed until twenty-four hours after the addition, while the expression was observed at forty-eight hours after the addition. These results suggest that the infection from the cell body side progresses differently to the infection from the synapse side. Next, we tried to optimize the four-dimensional imaging system for live fluorescent-cells. For long term culture under the microscope, we changed the medium into CO2 independent medium in a closed system. This enabled cells to live longer under the microscope. We tried various conditions of laser intensity, pinhole size, and number of Z slices for long term observation under the confocal laser microscope. However, we could not optimize the conditions for the long term observation as long as the confocal laser microscope was used.