Role of astrocytic remodeling in sleep/wake regulation

越来越多的证据表明，星形胶质细胞在包括突触可塑性在内的脑功能中发挥着重要作用。星形胶质细胞在几分钟内表现出其远端突起的快速重塑。星形胶质细胞的重塑可以发生在对稳态挑战的反应中，这些结构的变化可以改变突触的有效性和神经元的兴奋性。我们假设星形胶质细胞通过动态重塑突触周围突触过程来调节关键的睡眠/唤醒神经元，从而在睡眠/觉醒调节中发挥作用。我们将使用急性(6h)睡眠剥夺(SD)来延长大鼠的清醒时间，以及未受干扰和恢复的条件，并结合形态学(免疫组织化学、共聚焦显微镜)、电生理(膜片钳记录)和行为(EEG和EMG记录，星形胶质细胞功能的抑制物)技术。第一个形态研究将使用神经胶质谷氨酸转运蛋白-1(GLT-1)的免疫组织化学和共聚焦显微镜来分析SD或对照条件下6h后GLT-1与关键的睡眠/觉醒促进神经元的结合。初步结果表明，与时间匹配的对照组相比，SD后6h，下丘脑中促进觉醒的增食欲素(ORX)神经元和含睡眠的黑色素浓缩激素(MCH)神经元的星形胶质细胞GLT-1与其胞体结合的量呈现相反的变化。这些初步数据表明，星形细胞突起与睡眠/觉醒调节神经元的对位显示出动态变化，这取决于睡眠/觉醒历史以一种特定细胞类型的方式。星形胶质细胞定位的这种变化可以改变谷氨酸的摄取效率，从而改变突触的有效性和神经元的兴奋性。为了测试这种可能性，第二项研究将使用从睡眠剥夺或未受干扰的大鼠制备的脑片进行膜片钳记录。将研究基本的细胞膜特性和突触效能，以及SD的作用以及使用二氢多卡因(DHK)对GLT-1的药理阻断作用。我们用SD的初步数据显示ORX神经元的去极化，而不是MCH的去极化，以及SD对ORX神经元的DHK效应的阻断。这些初步的形态和电生理数据与星形胶质细胞可以重组以特定类型的细胞方式调节睡眠/唤醒调节神经元的概念一致。因此，第三项研究将利用大鼠的脑电和肌电记录，研究脑区特异性抑制星形细胞重塑(使用siRNA沉默星形细胞重塑的负面调节因子连接蛋白30的表达)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来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.