论文部分内容阅读
Rac1属于小G蛋白Rho GTPases家族的一员,通过调控细胞骨架及形态影响细胞迁移、轴突导向等生理过程,在脑内发挥重要作用。但Rac1的动态激活过程对脑内的生理学功能的影响尚不明确。近期报道可通过Rac1的光活化形式(photoactivatable Rac1,PA-Rac1)实现对Rac1活性的时空特异性操控。因此,我们构建并包装了由小鼠胶质原纤维酸性蛋白(mouse glial fibrillary acidic protein,m GFAP)启动子驱动的PA-Rac1及其光脱敏(light-insensitive)突变体PA-Rac1-C450A的腺相关病毒(adenoassociated virus,AAV),拟通过光刺激实时操控星形胶质细胞内Rac1的活性。我们将AAV-PA-Rac1及AAV-PA-Rac1-C450A感染原代星形胶质细胞。荧光实时成像实验结果显示光刺激表达PA-Rac1的原代星形胶质细胞时,在光刺激点附近表现出细胞膜的突起,而光刺激表达PA-Rac1-C450A的原代星形胶质细胞时,其细胞膜形态无明显改变。为进一步探究Rac1激活对小鼠关联性学习的影响,我们在小鼠背侧海马脑区注射AAV-PA-Rac1及AAV-PA-Rac1-C450A,在条件恐惧性记忆的训练过程中光激活背侧海马的Rac1。结果显示,表达PA-Rac1的小鼠在训练过程中的学习曲线显著低于对照组,而表达其突变体PA-Rac1-C450A组小鼠学习曲线和对照组相比无显著差异,表明光激活海马星形胶质细胞Rac1抑制了小鼠条件性恐惧记忆的形成。以上结果提示,背侧海马星形胶质细胞Rac1的激活参与小鼠的场景关联性学习过程。
Rac1, a member of the small G-protein Rho GTPases family, plays an important role in the brain through the regulation of cytoskeleton and morphological changes such as cell migration and axon guidance. However, the effect of dynamic activation of Rac1 on physiological functions in the brain is not yet clear. It has recently been reported that spatiotemporal manipulation of Racl activity can be achieved by photoactivatable Racl, PA-Racl. Thus, we constructed and packaged PA-Rac1 and its light-insensitive mutant PA-Rac1-C450A driven by the mouse glial fibrillary acidic protein (m GFAP) promoter Of adenoassociated virus (AAV), intended to manipulate Rac1 activity in astrocytes in real time by light stimulation. We infected AAV-PA-Rac1 and AAV-PA-Rac1-C450A with primary astrocytes. Fluorescence real-time imaging experiments showed that when light stimulated PA-Rac1-expressing primary astrocytes, protrusions of the cell membrane were shown in the vicinity of the light stimulus spot and light stimulation of primary astrocytes expressing PA-Rac1-C450A When the cell membrane morphology no significant change. To further investigate the effect of Rac1 activation on mouse learning and learning, we injected AAV-PA-Rac1 and AAV-PA-Rac1-C450A in the dorsal hippocampus of mice to activate light-activated dorsal Side of the seahorse Rac1. The results showed that the learning curve of PA-Rac1-expressing mice during training was significantly lower than that of the control group, while the mice learning the PA-Rac1-C450A mutant had no significant difference in learning curve compared with the control group, indicating that light-activated Rac1, a astrocyte in the hippocampus, inhibited the formation of conditioned fear memory in mice. The above results suggest that the activation of Rac1 in the dorsal hippocampal astrocytes is involved in the context-dependent learning process in mice.