Imaging Mitochondrial Function in Excitotoxicity

描述（由申请人提供）：与大多数其他器官系统相比，正常的脑功能需要不成比例的大能量供应，在许多神经退行性疾病中，即使是短暂的脑代谢中断也会导致认知或运动功能的灾难性丧失。缺血损伤可导致神经递质谷氨酸的不调节释放，并导致神经元不适当的过度兴奋，从而引发细胞死亡。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”，也可能涉及一系列疾病，包括癫痫发作、帕金森病和ALS。维持适当代谢功能的策略可能是设计未来兴奋性毒性损伤治疗干预措施的关键考虑因素。这种干预的成功依赖于对不同类型谷氨酸兴奋毒性所涉及的代谢需求的理解。本实验将在急性海马切片中评估线粒体功能，以评估谷氨酸受体刺激后线粒体功能原位改变的机制。用于研究线粒体功能的主要方法将是内在代谢信号的荧光成像，这种方法已在许多生化和一些成像研究中得到验证，但由于将高分辨率成像应用于完整制剂，它已重新引起人们的兴趣。在急性切片中使用成像方法可以在完整的准备中区分胶质和神经元代谢的贡献。内源性释放谷氨酸的反应（无论是在电去极化或缺氧/低血糖挑战期间）与谷氨酸受体亚型选择性激动剂的反应进行比较。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/成像将用于识别代谢变化的机制和细胞，以及选择性修改神经元与神经胶质代谢途径反应的药物干预。本征荧光研究将辅以线粒体内膜电位的荧光成像，以及导致代谢功能障碍的离子通量的单细胞电生理分析。由于海马CA1神经元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机制和兴奋性毒性细胞死亡机制的大量文献，海马CA1神经元将成为大多数研究的主题。为了研究短暂性缺血后死亡神经元的线粒体功能（Specific Aim 3），我们将利用短暂性前脑缺血沙鼠的制剂。

DESCRIPTION (provided by applicant): 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 over excitation 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 excitotoxicity. 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.