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Study on the effects of stressor on a organism using the technology in neuroscience

利用神经科学技术研究压力源对生物体的影响

基本信息

批准号：
12670313
负责人：
HOSOKAWA Toshiyuki
金额：
$ 2.18万
依托单位：
HOKKAIDO UNIVERSITY
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 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670313/
关键词：
stress hippocampus hormone synapse environmental edocrine disrupters コルチコイド

项目摘要

To study changes in synaptic morphology associated with steroid concentration, we visualized synaptic structures with high resolution imaging of confocal microscopy in hippocampal slices of adult rats. Synaptic structures are labeled by micro injection of a concentrated oil solution of Dil into the superfused slice as reported previously. Dye droplets are removed after 20 min. Estrogen and cortisol are exposed through ACSF solution or a dye ball. These sets of images were analyzed and compared in the dendritic spine density. No significant changes could be seen in the slices with exposure to high concentration of estrogen. Cortisol exposed slices showed significant reduction of spine density.We established organotypic culture using a filter. Confocal microscopy was applied to the slice to confirm the health of cells with propidium iodide. Optical recording to see the electrophysiological response on large area was applied and got good series of images which time resolution is 0.6 msec and spatial resolution is 128x128 pixels.We also studied the effect of TBT(tri-n-butyltin) that is one of the environmental endocrine disrupters to PC12 cell. It shows that TBT 10^<-12>-10^<-13> in concentration significantly reduced the ratio of apoptosis.The technique of electrophysiology (LTP:long-term potentiation) was applied to the hippocampal slices. Extracellular recording in the CA1 area showed that 5 μ M TBT decreased the slope of the EPSP (Excitatory Post-Synaptic Potential) significantly. It was studied at the early time (30 min after incubation of TBT). The induction of LTP needs NMDA receptor that is one kind of glutamate receptor. It means that NMDA receptor was damaged by the injection of TBT. It shows that if the concentration of TBT is higher it affects to the central nervous system.

为了研究与类固醇浓度相关的突触形态学变化，我们用高分辨率共聚焦显微镜在成年大鼠海马切片中观察突触结构。如前所述，通过将Dil的浓缩油溶液微量注射到灌流切片中来标记突触结构。20分钟后去除染料滴。通过ACSF溶液或染料球暴露雌激素和皮质醇。分析比较各组图像的树突棘密度。高浓度雌激素处理的切片未见明显变化。皮质醇暴露的切片显示棘密度显著降低。我们使用过滤器建立器官型培养。将共聚焦显微镜应用于切片以用碘化丙啶确认细胞的健康。采用光学记录技术观察大面积的电生理反应，获得了时间分辨率为0.6毫秒、空间分辨率为128 x128像素的良好图像系列，并研究了环境内分泌干扰物之一TBT（三丁基锡）对PC 12细胞的影响。结果表明，TBT 10^<-12>-10 ^<-13>能显著降低海马脑片细胞凋亡率。在CA 1区的细胞外记录显示，5 μ MTBT显著降低兴奋性突触后电位（EPSP）的斜率。在早期（TBT孵育后30 min）进行了研究。LTP的诱导需要谷氨酸受体之一的NMDA受体。说明TBT对NMDA受体有损伤作用。说明TBT浓度较高时会对中枢神经系统产生影响。