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，细胞 和大脑的化学环境。而细胞培养实验表明 星形胶质细胞的特性使它们能够影响 神经元的兴奋性和参与大脑的功能，有 几乎没有证据表明在体内扰动星形胶质细胞会影响大脑 功能。很有可能是因为缺乏支持 星形胶质细胞在脑中的功能作用源于缺乏方法 在体内研究星形胶质细胞。这项提议有两个主要目标。第一, 开发一种模型系统，使星形胶质细胞的特性可以 在体内系统地扰动使这些属性的作用 在大脑生理学方面可以被评估。其次，为了检验假设， 星形胶质细胞间缝隙连接通讯中断导致 神经细胞内细胞外K+浓度的升高及其伴随的浓度升高 兴奋性。 关于神经元-星形胶质细胞的一个更重要的假说 相互作用是星形胶质细胞通过其自身的 能够将细胞外K+维持在所需的狭窄范围内 对于正常的神经元活动来说。这一过程被称为空间 缓冲，被认为是通过吸收K+来完成的 内向整流钾通道及其向星形细胞的消散 由缝隙连接形成的合胞体。所有的迹象都那么小 脑细胞外[K+]升高显著增加神经元 兴奋性，这种兴奋性的增加会导致癫痫发作 活性和/或兴奋性毒性。 为了扰乱体内的星形细胞特性，DNA构建旨在 下调特定星形细胞基因产物的表达将 应用于海马区的CA1和CA3区 腺病毒和腺相关病毒载体。这些载体已经被 据报道，转导脑细胞具有高效和稳定的特点。 我们将重点研究海马体的放射层。 其中CA1和CA3锥体细胞树突接受兴奋性输入和 已知嵌入在星形细胞合胞体中。这些地区有 被广泛用于研究神经元兴奋性、长时程增强、惊厥活动 和兴奋性毒性。我们的长期目标是充分理解 与CA1和CA3S突触相关的星形胶质细胞在辐射中的作用 在正常和病理条件下。 四个可检验的假设将被检验。首先，腺病毒和/或 腺相关病毒载体可用于转导星形胶质细胞 活着。其次，这种基因结构可以用来消除GAP 体外连接通讯。第三，这种拆卸结构 放入腺病毒或腺相关病毒载体中，并 注射入海马区可减少体内的缝隙连接通讯。 第四，构建了拆卸缝隙连接通信 星形胶质细胞之间的相互作用会增加细胞外[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.