星形胶质细胞形态可塑性参与吸入麻醉药物的作用机制研究

How inhalation anesthetics modulate the consciousness state is poorly understood. Recent studies indicate that the state shift between anesthesia and wakefulness involves in changes of extracellular ions concentration and extracellular space (ECS) volume in the brain. Astrocytes mediate extracellular ion homeostasis and modulate the ECS volume, suggesting their potential roles in inhalation anesthetics-induced consciousness state shift. Based on recent advances including our pilot studies, we propose that inhalation anesthetics affect astrocyte morphological plasticity through a calcium-dependent mechanism, and astrocyte morphological plasticity participates in the changes of the ECS volume and extracellular ions concentration, thereby regulate neuronal excitability and consciousness state shift. By using astrocyte-specific genetic manipulation, in combined with iontophoresis, serial block face scanning electron microscopy, astrocyte confocal calcium signaling and remote telemetry, we will elucidate how inhalation anesthetics affect astrocyte morphological plasticity, and its dependence of astrocytes calcium signaling. By manipulating astrocyte morphology, we expect to change the ECS volume, extracellular ions concentration and neuronal excitability，further impact the inhalation anesthetics mediate consciousness state shift. The experiments in this propose would provide new insights into the action mechanisms of inhalation anesthetics and into the prevention strategies of post anesthesia-induced neurological disorders.

吸入麻醉药物对大脑意识的调控机制不明。最新研究发现麻醉与苏醒的意识转换与脑组织中细胞外离子浓度及细胞外间隙(ECS)容积动态变化有关。星形胶质细胞维持细胞外离子稳态，并参与ECS容积的调节，提示其有可能参与吸入麻醉药物介导的意识状态转换。基于前期研究基础，我们提出假说：吸入麻醉药物影响星形胶质细胞的形态可塑性，从而调节ECS容积和细胞外离子浓度，并影响神经元兴奋性和意识状态的转换。通过在体动物模型中的星形胶质细胞特异性基因编辑，采用离子导入法、连续块面电镜、钙成像和遥感生物电信号测定等技术，本课题将阐释吸入麻醉药物对星形胶质细胞形态可塑性的影响及钙信号在其中的作用，通过操控星形胶质细胞形态可塑性以调节ECS的容积、细胞外离子浓度和神经元兴奋性，进而影响吸入麻醉药介导的意识状态转换。本课题的实施将从胶质细胞的角度为麻醉机制研究和麻醉术后脑功能紊乱的防治提供新思路。

利用荧光蛋白对星形胶质细胞（星胶）及神经元进行稀疏标记，急性脑片或活体动物接受2.5%七氟烷（Sevo）暴露均能引起星胶精细分支形态缺失，伴随星胶-突触空间联系的弱化。免疫荧光的结果发现，原代培养的星胶或小鼠接受Sevo暴露，均能下调星胶磷酸化Ezrin（pEzrin）的水平，但均不影响Ezrin的总蛋白水平；通过腺相关病毒（AAV）将不能磷酸化的Ezrin突变体（EzrinT567A）过表达在小鼠皮质的星胶内，则能引起星胶形态缺失及星胶-突触空间联系弱化。利用γ-氨基丁酸（GABA）或谷氨酸的荧光探针（iGABASnFR或iGluSnFR）及活体成像，我们发现Sevo麻醉降低了WT小鼠细胞外GABA浓度，且EzrinT567A能进一步降低Sevo维持期间的细胞外GABA浓度，但不影响细胞外谷氨酸的水平。急性脑片电生理提示，EzrinT567A可增加Sevo给药后椎体神经元的Tonic GABA电流，但不影响其抑制性突触后电流（mIPSCs）的频率及幅度。EzrinT567A过表达及Ezrin条件性敲除均能增加Sevo的麻醉敏感性。我们的研究揭示了星胶形态可塑性通过调控星胶-突触空间联系，继而增加tonic GABA并参与吸入麻醉药的作用过程。