Astrocyte volume-regulated control of neuronal excitability

描述(申请人提供)：大量证据表明，星形胶质细胞积极参与调节神经元兴奋性，但星形胶质细胞肿胀在神经元兴奋性控制中的作用从未被直接测试过。我们的长期目标是识别和理解控制神经元兴奋性的星形细胞机制。这一特殊应用的目的是确定星形胶质细胞肿胀和肿胀引起的谷氨酸释放的特定操作如何在原位和体内导致神经元兴奋性的变化。中心假说是星形胶质细胞体积调节的阴离子通道(Vrac)中星形胶质细胞的肿胀和谷氨酸的释放是提高在体神经元兴奋性的必要条件和充分条件。这项研究的基本原理是，识别控制神经元兴奋性的新的星形细胞通路将为神经疾病和神经退行性疾病的治疗提供新的星形细胞药物靶点。在强大的初步数据的指导下，中心假说将通过追求三个具体目标来检验：1)确定星形胶质细胞肿胀引起的谷氨酸释放在多大程度上是增加神经元原位兴奋性的必要条件；2)确定星形胶质细胞肿胀引起的谷氨酸释放在多大程度上足以增加神经元的就地兴奋性；以及3)确定星形胶质细胞肿胀对体内神经元兴奋性控制的贡献。将使用膜片钳和转基因方法选择性地操纵星形胶质细胞肿胀和谷氨酸释放，并在记录急性海马片CA1区锥体神经元NMDA受体活性的过程中实时成像星形胶质细胞体积变化(目标1和2)，并将在体内分析低渗、高渗和选择性抑制剂对星形胶质细胞体积变化和神经元兴奋性的影响(目标3)。在我们看来，我们的方法是创新的，因为它与评估星形胶质细胞钙依赖的胶质传递在调节神经元兴奋性中的作用的现状有很大的不同，因为我们已经开发出并被证明是可行的技术，可以选择性和特异性地操纵星形胶质细胞的体积变化和谷氨酸的释放。这项拟议的研究意义重大，因为一旦星形胶质细胞控制神经元兴奋性的机制得到澄清，就可以设计出新的星形胶质细胞导向疗法来防止过度的神经元兴奋性，同时保持神经元兴奋性的基础水平和正常认知功能不变。这些知识还将为治疗与细胞体积变化(包括各种形式的水肿)相关的神经系统疾病提供新的策略，同时也从根本上促进我们对神经胶质-神经元相互作用的理解。

DESCRIPTION (provided by applicant): Numerous lines of evidence suggest that astrocytes actively participate in regulating neuronal excitability, but the role of astrocyte swelling in conrol of neuronal excitability has never been directly tested. Our long-term goal is to identify and understand astrocytic mechanisms controlling neuronal excitability. The objective in this particular application is to determine how specific manipulations of astrocyte swelling and swelling-evoked glutamate release lead to changes in neuronal excitability in situ and in vivo. The central hypothesis is that astrocyte swelling and glutamate release from astrocytic volume-regulated anion channels (VRAC) is both necessary and sufficient to elevate neuronal excitability in situ and in vivo. The rationale for the proposed research is that, identification o novel astrocytic pathways controlling neuronal excitability will provide new astrocytic drug targets for the treatment of neurological disorders and neurodegenerative disease. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the extent to which astrocyte swelling-evoked glutamate release is necessary to increase neuronal excitability in situ; 2) Determine the extent to which astrocyte swelling-evoked glutamate release is sufficient to increase neuronal excitability in situ; and 3) Determine the contribution of astrocyte swelling to the control of neuronal excitability in vivo. Astrocyte swellng and glutamate release will be selectively manipulated using patch clamp and transgenic approaches, together with real-time imaging of astrocyte volume changes during recording of NMDA receptor activity in CA1 pyramidal neurons in acute hippocampal slices (Aims 1 and 2), and the effects of hypoosmolarity, hyperosmolarity and selective inhibitors on astrocytic volume changes and neuronal excitability will be assayed in vivo (Aim 3). Our approach is innovative, in our opinion, because it represents a significant departure from the status quo of assessing the role of astrocyte Ca2+-dependent gliotransmission in regulating neuronal excitability, and because techniques have been developed and proven feasible in our hands to selectively and specifically manipulate astrocyte volume changes and release of glutamate. The proposed re- search is significant, because once astrocytic mechanisms controlling neuronal excitability become clarified, novel astrocyte-directed therapies can be devised to prevent excessive levels of neuronal excitability while leaving basal levels of neuronal excitability and normal cognitive function intact. Such knowledge will also pro- vide new strategies to treat neurological disorders associated with cellular volume changes (including various forms of edema), while also fundamentally advancing our understanding of glial-neuronal interactions.