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光激活背侧海马星形胶质细胞Rac1抑制小鼠关联性记忆的形成

郭潇木, 廖朝晖, 陶冶铮, 王菲菲, 马兰^*

复旦大学基础医学院医学神经生物学国家重点实验室，药理研究中心，上海 200032

摘要

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, mGFAP)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(adeno- associated 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。

关键词： 光激活Rac1; 星形胶质细胞 ; 光激活 ; 关联性学习

分类号：R964

Optogenetic activation of dorsal hippocampal astrocytic Rac1 blocks the learning of associative memory

GUO Xiao-Mu, LIAO Zhao-Hui, TAO Ye-Zheng, WANG Fei-Fei, MA Lan^*

The State Key Laboratory of Medical Neurobiology and Pharmacology Research Center, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China

Abstract

Rac1 belongs to the family of Rho GTPases, and plays important roles in the brain function. It affects the cell migration and axon guidance via regulating the cytoskeleton and cellular morphology. However, the effect of its dynamic activation in regulating physiological function remains unclear. Recently, a photoactivatable analogue of Rac1 (PA-Rac1) has been developed, allowing the activation of Rac1 by the specific wavelength of light in living cells. Thus, we constructed recombinant adeno-associated virus (AAV) of PA-Rac1 and its light-insensitive mutant PA-Rac1-C450A under the control of the mouse glial fibrillary acidic protein (mGFAP) promoter to manipulate Rac1 activity in astrocytes by optical stimulation. Primary culture of hippocampal astrocytes was infected with the recombinant AAV-PA-Rac1 or AAV-PA-Rac1-C450A. Real-time fluorescence imaging showed that the cell membrane of the astrocyte expressing PA-Rac1 protruded near the light spot, while the astrocyte expressing PA-Rac1-C450A did not. We injected AAV-PA-Rac1 and AAV-PA-Rac1-C450A into dorsal hippocampus to investigate the role of the activation of Rac1 in regulating the associative learning. With optical stimulation, the PA-Rac1 group, rather than the PA-Rac1-C450A group, showed slower learning curve during the fear conditioning compared with the control group, indicating that activating astrocytic Rac1 blocks the formation of contextual memory. Our data suggest that the activation of Rac1 in dorsal hippocampal astrocyte plays an important role in the associative learning.

Key words: photoactivatable analogue of Rac1; astrocyte ; optogenetic activation ; associative learning

收稿日期：2017-02-24　　录用日期：2017-03-28

通讯作者：马兰　　E-mail: lanma@fudan.edu.cn

引用本文：

郭潇木, 廖朝晖, 陶冶铮, 王菲菲, 马兰. 光激活背侧海马星形胶质细胞Rac1抑制小鼠关联性记忆的形成[J]. 生理学报 2017; 69 (3): 241-251.

GUO Xiao-Mu, LIAO Zhao-Hui, TAO Ye-Zheng, WANG Fei-Fei, MA Lan. Optogenetic activation of dorsal hippocampal astrocytic Rac1 blocks the learning of associative memory. Acta Physiol Sin 2017; 69 (3): 241-251 (in Chinese with English abstract).