Astrocyte volume-regulated control of neuronal excitability

描述（由申请人提供）：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控制神经元兴奋性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胶质细胞机制。在这个特定的应用程序的目的是确定如何特定的操作星形胶质细胞肿胀和肿胀诱发的谷氨酸释放导致神经元兴奋性的变化，在原位和体内。中心假设是星形胶质细胞肿胀和谷氨酸从星形胶质细胞体积调节阴离子通道（VRAC）的释放是必要的和足够的，以提高神经元的兴奋性在原位和体内。提出的研究的基本原理是，鉴定控制神经元兴奋性的新型星形胶质细胞通路将为治疗神经系统疾病和神经退行性疾病提供新的星形胶质细胞药物靶点。在强有力的初步数据的指导下，将通过追求三个具体目标来检验中心假设：1）确定星形胶质细胞肿胀诱发的谷氨酸释放在原位增加神经元兴奋性所必需的程度; 2）确定星形胶质细胞肿胀诱发的谷氨酸释放在原位足以增加神经元兴奋性的程度;和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，以及在记录急性海马切片中CA 1锥体神经元中NMDA受体活性期间星形胶质细胞体积变化的实时成像（目的1和2），并将在体内测定低渗透压、高渗透压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（目的3）。我们的方法是创新的，在我们看来，因为它代表了一个显着的偏离现状评估的作用星形胶质细胞钙依赖性gliotransmission调节神经元的兴奋性，因为技术已经开发和证明可行的，在我们手中选择性和特异性地操纵星形胶质细胞的体积变化和释放谷氨酸。所提出的研究是重要的，因为一旦控制神经元兴奋性的星形胶质细胞机制变得清晰，就可以设计新的星形胶质细胞定向疗法来防止神经元兴奋性的过度水平，同时保持神经元兴奋性和正常认知功能的基础水平不变。这些知识还将提供治疗与细胞体积变化相关的神经系统疾病（包括各种形式的水肿）的新策略，同时也从根本上推进了我们对神经胶质-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.