Imaging Mitochondrial Function in Excitotoxicity

与大多数其他器官系统相比，正常的大脑功能需要不成比例的大量能量。 供应，甚至大脑代谢的短暂中断都可能导致认知或 运动功能在广泛的神经退行性疾病。缺血性损伤可导致不受调节的 释放神经递质谷氨酸，并导致神经元过度兴奋， 引发细胞死亡谷氨酸受体过度激活后的细胞损伤过程 被称为“兴奋性毒性”，并且也可能涉及一系列疾病，包括癫痫发作活动， 帕金森病和ALS维持适当的代谢功能的策略可能是关键的 考虑设计未来的兴奋性毒性损伤的治疗干预措施。这样的成功 干预依赖于理解参与不同类型谷氨酸兴奋毒性的代谢需求。 本提案中的实验将评估急性海马切片中的线粒体功能，以评估 参与谷氨酸受体刺激后线粒体功能原位变化的机制。一 用于研究线粒体功能的主要方法将是内源性代谢的荧光成像 信号，这种方法已经在许多生物化学和一些成像研究中得到验证，但 由于高分辨率成像技术在无损检测中的应用， 准备工作在急性切片中使用成像方法允许神经胶质和神经元的贡献 在完整的制剂中区分代谢。对内源性释放谷氨酸的反应 （在电去极化或缺氧/低血糖激发期间）与响应进行比较 谷氨酸受体亚型选择性激动剂。单光子和双光子成像将用于识别 线粒体信号的细胞来源，单细胞电生理学/成像，以确定机制， 负责代谢变化的细胞和选择性改变代谢的药理学干预 神经元与神经胶质的反应通路。内在荧光研究将补充荧光 线粒体内膜电位成像和单细胞电生理学分析， 导致代谢功能障碍的通量。海马CA 1神经元将成为大多数研究的主题， 由于它们对兴奋性毒性损伤的敏感性以及关于 海马锥体神经元生理学和兴奋性毒性细胞死亡机制。为研究 线粒体功能的神经元注定死亡后短暂缺血（具体目标3），我们将利用 来自经历短暂前脑缺血的沙鼠的制剂。

Compared to most other organ systems, normal brain function requires a disproportionately large energy supply, and even transient disruption of brain metabolism can contribute to catastrophic loss of cognitive or motor function in a wide range of neurodegenerative disorders. Ischemic insults can lead to unregulated release of the neurotransmitter glutamate, and lead to inappropriate overexcitation of neurons to the point of triggering cell death. The process of cell damage following excessive glutamate receptor activation has been termed "excitotoxicity", and may also be involved in a range of disorders including seizure activity, Parkinson's Disease and ALS. Strategies that maintain appropriate metabolic function may be a critical consideration for the design of future therapeutic interventions for excitotoxic injuries. The success of such interventions relies on understanding metabolic demands involved in different types of glutamate excitoxicity. Experiments in this proposal will evaluate mitochondrial function in acute hippocampal slices, to evaluate the mechanisms involved in mitochondrial function changes in situ, following glutamate receptor stimulation. A major approach used to study mitochondrial function will be fluorescence imaging of intrinsic metabolic signals, an approach which has been validated in many biochemical and some imaging studies, but which has received a resurgence of interest because of the application of high resolution imaging to intact preparations. The use of imaging approaches in acute slices allows the contributions of glial and neuron metabolism to be differentiated in intact preparations. Responses to endogenously-released glutamate (either during electrical depolarization or hypoxic/hypoglycemic challenges) to be compared with responses to glutamate receptor subtype-selective agonists. Single- and 2-photon imaging will be used to identify cellular sources of mitochondrial signals, single cell electrophysiology/imaging to identify mechanisms and cells responsible for metabolic changes and pharmacological interventions that selectively modify metabolic pathways responses in neurons vs glia. Intrinsic fluorescence studies will be complemented by fluorescence imaging of mitochondrial inner membrane potential, and single cell electrophysiological analysis of ionic fluxes contributing to metabolic dysfunction. Hippocampal CA1 neurons will be the subject of most studies, because of their sensitivity to excitotoxic damage and the extensive literature on mechanisms of hippocampal pyramidal neuron physiology and mechanisms of excitotoxic cell death. For studies of mitochondrial function in neurons destined to die following transient ischemia (Specific Aim 3), we will utilize preparations from gerbils subjected to transient forebrain ischemia.