ASTROCYTIC REGULATION OF NEURONAL EXCITABILITY IN VIVO

我们坚信如果我们要了解星形胶质细胞的作用 在大脑中，这些细胞必须在复杂的几何结构中进行研究， 和化学环境。虽然细胞培养实验表明 星形胶质细胞具有能够影响 神经元兴奋性和参与大脑功能，有 几乎没有证据表明，在体内扰乱星形胶质细胞会影响大脑 功能很有可能，缺乏信息支持 星形胶质细胞在大脑中的功能作用源于缺乏方法， 研究体内的星形胶质细胞这项建议有两个主要目标。 第一、 开发一个模型系统， 在体内系统地扰动，使得这些性质的作用 大脑生理学可以评估。第二，为了检验假设， 星形胶质细胞之间缝隙连接通讯的中断导致 细胞外K+的增加和伴随的神经细胞内K+的增加， 兴奋性 关于神经元星形胶质细胞的一个重要假说 星形胶质细胞通过它们的相互作用来调节神经元的活动。 维持细胞外K+在所需的狭窄范围内的能力 正常的神经元活动这个过程被称为空间 缓冲，并被认为是通过吸收K+通过 内向整流钾通道及其向星形胶质细胞的耗散 由缝隙连接形成的合胞体。 所有迹象都表明 脑细胞外[K+]的增加显著增加神经元 这种兴奋性的增加会导致癫痫发作 活性和/或兴奋性毒性。 为了扰乱体内星形胶质细胞的特性，设计了DNA构建体， 敲低特定星形胶质细胞基因产物的表达， 将其递送到海马体的CA 1和CA 3区域， 腺病毒和腺相关病毒载体。这些带菌者 据报道，以高效率和稳定性来刺激脑细胞。 我们的研究将集中在海马体的辐射底层 其中CA 1和CA 3锥体细胞树突接收兴奋性输入， 被嵌入在星形胶质细胞合胞体中 这些地区 广泛用于研究神经元兴奋性、LTP、癫痫发作活动 和兴奋性毒性。我们的长期目标是充分理解 星形胶质细胞与CA 1和CA 3的突触有关。辐射性游戏 在正常和病理条件下。 将检验四个可检验的假设。 首先，腺病毒和/或 腺相关病毒载体可用于转染星形胶质细胞， vivo.第二，基因构建体可用于敲低gap， 体外连接通讯 第三， 置于腺病毒或腺相关病毒载体中， 注射到海马体中减少了体内间隙连接通讯。 第四，构建敲除的缝隙连接通讯 星形胶质细胞之间将增加细胞外[K+]和神经元 在原位和体内的兴奋性。

It is our conviction that if we are to understand the role of astrocytes in brain, these cells must be studied in the complex geometry, cellular and chemical milieu of brain. While cell culture experiments indicate that astroglia have properties that would enable them to influence neuronal excitability and participate in brain function, there is virtually no evidence that perturbing astrocytes in vivo affects brain function. It is very likely that the void of information supporting a functional role for astrocytes in brain stems from a lack of methods for studying astrocytes in vivo. This proposal has two major goals. First, to develop a model system whereby the properties of astrocytes can be systematically perturbed in vivo such that the role of these properties in brain physiology can be assessed. Second, to test the hypothesis that disruption of gap junction communication between astrocytes leads to increases in extracellular K+ and attendant increases in neuronal excitability. One of the more important hypotheses concerning neuronal-astrocyte interactions is that astrocytes regulate neuronal activity through their ability to maintain extracellular K+ within the narrow limits required for normal neuronal activity. This process is referred to as spatial buffering and is thought to be accomplished by the uptake of K+ through inward rectifying K+ channels and its dissipation into an astrocytic syncytium formed by gap junctions. All indications are that small increases in extracellular [K+] in brain markedly increase neuronal excitability and that this increase in excitability can lead to seizure activity and/or excitotoxicity. To perturb astrocytic properties in vivo, DNA constructs designed to knock-down the expression of specific astrocytic gene products will be delivered into the CA1 and CA3 regions of the hippocampus using adenoviral and adeno-associated viral vectors. These vectors have been reported to transduce brain cells with high efficiency and stability. We will focus our studies in the substratum radiatum of the hippocampus where CA1 and CA3 pyramidal cell dendrites receive excitatory input and are known to be embedded in an astrocyte syncytium. These regions have been used extensively to study neuron excitability, LTP, seizure activity and excitotoxicity. Our long-term goal is to fully understand the role that astrocytes associated with synapses in CA1 and CA3 s. radiatum play under normal and pathological conditions. Four testable hypotheses will be examined. First, that adenoviral and/or adeno-associated viral vectors can be used to transduce astrocytes in vivo. Second, that gene constructs can be used to knockdown gap junctional communication in vitro. Third, that knock-down constructs placed into either adenoviral or adeno-associated viral vectors and injected into the hippocampus reduce gap junction communication in vivo. And fourth, that constructs which knock-down gap junction communication between astrocytes will increase extracellular [K+]and neuronal excitability in situ and in vivo.