Imaging Mitochondrial Function in Excitotoxicity

兴奋性毒性中线粒体功能的成像

基本信息

批准号：
7031935
负责人：
Claude W Shuttleworth
金额：
$ 26.31万
依托单位：
UNIVERSITY OF NEW MEXICO
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2009-12-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：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，甚至大脑新陈代谢的短暂破坏也会导致多种神经退行性疾病中认知或运动功能的灾难性丧失。缺血性损伤会导致神经递质谷氨酸的释放不受控制，并导致神经元过度兴奋，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”，也可能与一系列疾病有关，包括癫痫发作、帕金森病和肌萎缩侧索硬化症。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。此类干预措施的成功取决于对不同类型谷氨酸兴奋性毒性涉及的代谢需求的了解。该提案中的实验将评估急性海马切片中的线粒体功能，以评估谷氨酸受体刺激后线粒体功能原位变化所涉及的机制。用于研究线粒体功能的主要方法是内在代谢信号的荧光成像，该方法已在许多生化和一些成像研究中得到验证，但由于高分辨率成像应用于完整制剂，该方法重新受到人们的关注。在急性切片中使用成像方法可以在完整的制剂中区分神经胶质和神经元代谢的贡献。对内源性释放的谷氨酸的反应（在电去极化或缺氧/低血糖挑战期间）与对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，单细胞电生理学/成像将用于识别负责代谢变化的机制和细胞，以及选择性改变神经元与神经胶质细胞代谢途径反应的药理学干预措施。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学分析将补充固有荧光研究。海马 CA1 神经元将成为大多数研究的主题，因为它们对兴奋性毒性损伤敏感，并且关于海马锥体神经元生理学机制和兴奋性毒性细胞死亡机制的大量文献。为了研究短暂性缺血后注定死亡的神经元的线粒体功能（具体目标 3），我们将使用来自遭受短暂前脑缺血的沙鼠的制剂。