Role of astrocytic remodeling in sleep/wake regulation

越来越多的证据表明星形胶质细胞在脑功能中发挥重要作用，包括突触可塑性。星形胶质细胞在几分钟内表现出其远端突起的快速重塑。星形胶质细胞重塑可以发生在对稳态挑战的反应中，这些结构变化可以改变突触的功效和神经元的兴奋性。我们假设星形胶质细胞通过动态重塑其突触周围过程来调节关键的睡眠/觉醒神经元，从而在睡眠/觉醒调节中发挥作用。我们将在大鼠中使用急性（6小时）睡眠剥夺（SD）来延长清醒时间，同时在不受干扰和恢复的情况下，结合形态学（免疫组织化学，共聚焦显微镜），电生理（膜片钳记录）和行为（脑电图和肌电图记录，星形胶质细胞功能抑制剂）技术。***第一次形态学研究将采用免疫组织化学方法检测神经胶质谷氨酸转运蛋白-1 (GLT-1)，并使用共聚焦显微镜分析在SD或对照条件下6小时后GLT-1与促进睡眠/觉醒的关键神经元的关联。初步结果表明，相对于时间匹配的对照组，下丘脑中含有促醒食欲素（ORX）的神经元和含有促睡眠黑色素集中激素（MCH）的神经元在SD 6小时后与其细胞体的星形胶质细胞GLT-1的含量发生了相互变化。这些初步数据表明，星形细胞过程与睡眠/觉醒调节神经元的关系以一种细胞类型特异性的方式根据睡眠/觉醒历史发生动态变化。星形胶质细胞的这种变化可以改变谷氨酸摄取效率，从而改变突触的功效和神经元的兴奋性。为了测试这种可能性，第二项研究将使用膜片钳记录睡眠不足或未受干扰的大鼠的大脑切片。本研究将探讨GLT-1的基本膜特性和突触效应，并研究SD的作用以及二氢海碱盐（DHK）对GLT-1的药物阻断作用。我们使用SD的初步数据显示ORX神经元去极化，但MCH神经元没有去极化，并且SD在ORX神经元中阻断了DHK效应。这些初步形态学和电生理数据与星形胶质细胞可以重组以细胞类型特异性方式调节睡眠/觉醒调节神经元的观点一致。因此，第三项研究将利用大鼠脑电图和肌电图记录，研究脑区域特异性抑制星形细胞重构（使用siRNA沉默连接蛋白30的表达，星形细胞重构的负调节因子）对睡眠/觉醒模式和SD后睡眠反弹的影响。这项拟议的研究有望为星形胶质细胞如何与神经元相互作用以调节睡眠和清醒状态提供新的见解。

Increasing evidence indicates that astrocytes play important roles in brain functions, including synaptic plasticity. Astrocytes demonstrate rapid remodeling of their distal processes over the course of minutes. Astrocytic remodeling can occur in response to homeostatic challenges, and these structural changes can alter synaptic efficacy and neuronal excitability. We hypothesize that astrocytes play a role in sleep/wake regulation through dynamic remodeling of their perisynaptic processes to regulate key sleep/wake-promoting neurons. We will use acute (6 h) sleep deprivation (SD) for prolonged wakefulness, along with undisturbed and recovery conditions, in conjunction with morphological (immunohistochemistry, confocal microscopy), electrophysiological (patch-clamp recording), and behavioural (EEG & EMG recording, inhibitors of astrocyte functions) techniques in rats. ***The first morphological study will use immunohistochemistry for glial glutamate transporter-1 (GLT-1) and confocal microscopy to analyze GLT-1 apposition with key sleep/wake-promoting neurons after 6 h of SD or control conditions. Preliminary results indicate that wake-promoting orexin (ORX)-containing neurons, and sleep-promoting melanin concentrating hormone (MCH)-containing neurons in the hypothalamus show reciprocal changes in the amount of astrocytic GLT-1 apposition with their cell bodies after 6 h of SD, relative to time-matched controls. These preliminary data suggest that astrocytic processes show dynamic changes in their apposition with sleep/wake-regulatory neurons depending on sleep/wake history in a cell type-specific manner. Such changes in astrocytic apposition can alter glutamate uptake efficiency, and thereby synaptic efficacy and neuronal excitability. To test this possibility, the second study will use patch-clamp recording from brain slices prepared from sleep-deprived or undisturbed rats. Basic membrane properties and synaptic efficacy will be investigated, and effects of SD as well as pharmacological blockade of GLT-1 using dihydorkainate (DHK) will be characterized. Our preliminary data with SD show depolarization of ORX, but not MCH, neurons, and occlusion of the DHK effects by SD in ORX neurons. These preliminary morphological and electrophysiological data are consistent with the notion that astrocytes can restructure to modulate sleep/wake regulatory neurons in a cell type-specific manner. Thus, the third study will examine the effect of brain region-specific inhibition of astrocytic remodeling (using siRNA to silence the expression of connexin 30, a negative regulator of astrocytic restructuring) on sleep/wake patterns, and on sleep rebound after SD, using EEG & EMG recordings in rats. The proposed research is expected to provide novel insights about how astrocytes interact with neurons to regulate sleep and wake states.