Neurons and Glia in vitro

描述(申请人提供)：新出现的证据表明，缝隙连接半通道(HCS)可以作为星形胶质细胞中的独立通道。缝隙连接和HCS的作用从根本上是不同的，星形胶质细胞HCS的生物学还知之甚少。与连接两个细胞内部的缝隙连接不同，开放的HC直接连接细胞内和细胞外空间。拟议中的实验将探索星形胶质细胞中这些通道的特性，并开始严格测试它们如何参与大脑中的正常和病理事件。将检验以下假设：1)星形胶质细胞表达功能性HCs。2)HC受多种因素的调控，包括二价阳离子、pH、膜电位、谷氨酸受体刺激、先前的HC活性和环境。3)HCs影响谷氨酸、离子和其他小分子的跨膜梯度。HCs在强烈的神经活动时开放，可能参与正常的大脑功能。4)Hcs在缺血时被激活，并参与了这种情况的病理生理过程。这些实验将使用原代培养的星形胶质细胞、脑片和一种有利的CMS白质准备，即急性分离的小鼠视神经。组织将从野生型和转基因小鼠身上获得，这些小鼠不表达在星形胶质细胞中形成HCS的特定蛋白。将使用离子成像、染料摄取、高效液相氨基酸测量和免疫细胞化学来研究星形胶质细胞HCS的行为。建议的研究将提供关于星形胶质细胞HCS的有用的新信息，这是一个被忽视的具有独特特征的通道。这些结果将对正常和病理的大脑功能都有影响。

DESCRIPTION (provided by applicant): Emerging evidence suggests that gap junction hemichannels (HCs) can act as stand alone channels in astrocytes. The actions of gap junctions and HCs are fundamentally different and the Biology of astrocyte HCs is poorly understood. Unlike gap junctions, which connect the interiors of two cells, open HCs directly connect intracellular and extracellular spaces. The proposed experiments will explore the properties of these channels in astrocytes and begin the process of critically testing how they participate in both normal as well as pathological events in the brain. The following hypotheses will be tested: 1) Astrocytes express functional HCs. 2) HCs are gated and/or modulated by multiple factors including divalent cations, pH, membrane potential, glutamate receptor stimulation, prior HC activity and environment. 3) HCs influence transmembrane gradients of glutamate, ions and other small molecules. HCs open during intense neural activity and may participate in normal brain functions. 4) HCs are activated during ischemia and contribute to the pathophysiology of this condition. These experiments will use primary cultured astrocytes, brain slices and an advantageous CMS white matter preparation, the acutely isolated mouse optic nerve. Tissue will be obtained from wild type and from genetically altered mice that do not express specific proteins that form HCs in astrocytes. The behavior of astrocyte HCs will be studied using ion imaging, dye uptake, HPLC amino acid measurements and immunocytochemistry. The proposed studies will provide useful new information about astrocyte HCs, a neglected channel with unique characteristics. These results will have implications for both normal as well as pathological brain function.