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能量供应，甚至脑部代谢的短暂破坏也会导致多种神经退行性疾病的认知或运动功能的灾难性丧失。缺血性侮辱会导致神经递质谷氨酸的不受监管的释放，并导致对神经元激发的不适当，直到触发细胞死亡。过度谷氨酸受体激活后细胞损伤的过程称为“兴奋性毒性”，也可能参与包括癫痫活性，帕金森氏病和ALS在内的一系列疾病。维持适当代谢功能的策略可能是设计对兴奋性毒性损伤的未来治疗干预措施的关键考虑。这种干预措施的成功依赖于理解不同类型的谷氨酸兴奋性毒性所涉及的代谢需求。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的线粒体功能变化所涉及的机制。一种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，这种方法已在许多生化和一些成像研究中得到了验证，但是由于将高分辨率成像应用于完整的制剂，因此人们引起了人们的兴趣。急性切片中的成像方法的使用使神经胶质和神经元代谢的贡献在完整的制剂中有所区分。将对内源性发行的谷氨酸的反应（在去极化或低氧/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。单个和2光子成像将用于鉴定线粒体信号的细胞来源，单细胞电生理学/成像，以鉴定负责代谢变化和药理干预措施的机制和细胞，以选择性地改变神经元与Glia中的代谢途径反应。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，固有的荧光研究将得到补充。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的敏感性以及有关海马锥体神经元生理机制的广泛文献以及兴奋性细胞死亡的机制。为了研究一定会在短暂性缺血后死亡的神经元中的线粒体功能（特定目标3），我们将利用受到短暂前脑缺血的球鼠的制剂。