Astrocyte volume-regulated control of neuronal excitability

描述（申请人提供）：许多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星形细胞控制神经元兴奋性的机制。这个特殊应用的目的是确定星形胶质细胞肿胀和肿胀诱发谷氨酸释放的特定操作如何导致原位和体内神经元兴奋性的变化。核心假设是星形胶质细胞肿胀和谷氨酸释放从星形胶质细胞体积调节阴离子通道（VRAC）是必要和充分的，以提高神经元兴奋性在原位和体内。本研究的基本原理是，发现控制神经元兴奋性的星形细胞通路将为神经系统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。在强有力的初步数据的指导下，中心假设将通过追求三个具体目标来验证：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原位神经元兴奋性所必需的；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原位增加神经元的兴奋性；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。利用膜片钳和转基因方法选择性地控制星形胶质细胞的肿胀和谷氨酸释放，同时在记录急性海马切片CA1锥体神经元NMDA受体活性时实时成像星形胶质细胞体积变化（目的1和2），并在体内检测低渗、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（目的3）。在我们看来，我们的方法是创新的，因为它代表了评估星形胶质细胞Ca2+依赖性胶质传递在调节神经元兴奋性中的作用的现状的重大偏离，并且因为我们已经开发并证明了选择性地和特异性地操纵星形胶质细胞体积变化和谷氨酸释放的技术是可行的。这项研究意义重大，因为一旦星形胶质细胞控制神经元兴奋性的机制被阐明，新的星形胶质细胞导向的治疗方法就可以被设计出来，以防止神经元兴奋性水平过高，同时保持神经元兴奋性的基础水平和正常的认知功能不变。这些知识也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，同时也从根本上推进我们对胶质-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.