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。星形胶质细胞在数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响应于稳态挑战而发生，并且这些结构变化可以改变突触功效和神经元兴奋性。我们假设星形胶质细胞通过动态重塑突触周围过程来调节关键的睡眠/觉醒促进神经元，从而在睡眠/觉醒调节中发挥作用。我们将使用急性（6小时）睡眠剥夺（SD）延长清醒，沿着不受干扰和恢复条件，结合形态学（免疫组织化学，共聚焦显微镜），电生理学（膜片钳记录）和行为学（EEG和EMG记录，星形胶质细胞功能抑制剂）技术在大鼠中。* 第一项形态学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分析SD或对照条件6小时后GLT-1与关键睡眠/觉醒促进神经元的并置。初步结果表明，唤醒促进食欲素（ORX）的含神经元，和睡眠促进黑色素浓集激素（MCH）的含神经元在下丘脑显示相互的变化量的星形胶质细胞GLT-1沉积与他们的细胞体后6小时的SD，相对于时间匹配的控制。这些初步的数据表明，星形胶质细胞的进程显示动态变化，其与睡眠/觉醒调节神经元的并置依赖于睡眠/觉醒的历史，在细胞类型特异性的方式。星形胶质细胞并置的这种变化可以改变谷氨酸摄取效率，从而改变突触功效和神经元兴奋性。为了测试这种可能性，第二项研究将使用膜片钳记录从睡眠剥夺或不受干扰的大鼠制备的脑切片。将研究基本膜特性和突触功效，并表征SD的作用以及使用二氢红藻氨酸盐（DHK）对GLT-1的药理学阻断。我们的初步数据与SD显示ORX，但不是MCH，神经元的去极化，和闭塞的DHK的影响SD在ORX神经元。这些初步的形态学和电生理学数据与星形胶质细胞可以以细胞类型特异性方式重构以调节睡眠/觉醒调节神经元的概念一致。因此，第三项研究将使用大鼠的EEG和EMG记录来检查脑区域特异性抑制星形胶质细胞重构（使用siRNA来沉默连接蛋白30（星形胶质细胞重构的负调节剂）的表达）对睡眠/觉醒模式和SD后睡眠反弹的影响。这项拟议中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觉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.