Quantitation of Simultaneous Hydrogen Peroxide and Dopamine Dynamics In Vivo

描述（由申请人提供）：在理解氧化损伤如何促进发病机制方面存在根本性差距。 因此，长期目标是阐明大脑中几种活性氧和小分子的释放/清除动力学如何成为涉及氧化应激的神经退行性疾病状态的基础。 过氧化氢（H2 O2）是一种活性氧，也是正常脑功能中的重要信号分子。 由于H2 O2具有这些不同的生物学作用，因此H2 O2浓度可能以精确的空间和时间分辨率在细胞外空间中上升和下降，使得可以实现信号传导的功能水平，同时防止由不受调节的产生引起的病理后果。 然而，旨在阐明这些动力学的研究受到了阻碍，缺乏一种方法来探测动态H2 O2波动在生活系统中的分子特异性。 本研究的目标是使内源性H2 O2波动的定量分析实时，并阐明这些分子动力学如何调节多巴胺（DA）在完整的功能性脑组织。 H2 O2参与帕金森病的发病机制。 同时H2 O2和DA的测量将使监管动力学和机制被解开，这些机制的疾病或药物的改变调查，并澄清运动功能障碍的神经化学过程。 碳纤维微电极将与快速扫描循环伏安法一起使用，因为这种方法提供了实时离散大脑位置神经传递的定量视图。 具体目标是联合收割机将新技术的开发与创新应用相结合。 分别是：1. 为了使H2 O2波动在特定的脑细胞核的细胞外空间的精确表征，阐明其调节信号作用，突触外寿命，影响范围，和正常和病理条件下的扩散曲线。 这些实验还将证明H2 O2的各种来源在多大程度上有助于选择脑核团内的信号传导。 2. 阐明H2 O2和DA之间精确的生理相互作用，以及这些分子动力学在帕金森病相关运动并发症发病中的作用。 为了实现这些目标，将开发和验证功能强大的数学模型，可用于解释药物对H2 O2生成和清除之间平衡的影响。 将对现有分析技术进行修改，以便在化学品多变的情况下改进定量评估。 这项研究意义重大，因为其结果有望纵向推进和扩展我们对H2 O2在大脑中所起生理作用的理解，并阐明氧化应激是否是多巴胺能功能障碍的引发者，或者是该过程的结果。 最终，这些知识将为基于氧化还原生物学的改进的治疗干预、神经保护策略和有前途的抗帕金森病药物的开发提供信息。 公共卫生相关性：拟议的研究与公共卫生有关，因为表征H2 O2在大脑中的波动，并阐明H2 O2和多巴胺之间的精确生理相互作用，预计将有助于确定这些分子动力学在帕金森病相关运动并发症发病中的作用。 因此，拟议的研究与NIH的使命的一部分有关，即发展有助于改善健康和减少疾病负担的基础知识。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how oxidative damage contributes to pathogenesis. Thus, the long-term goal is to elucidate how the release/clearance dynamics of several reactive oxygen species and small molecules in the brain underlie neurodegenerative disease states involving oxidative stress. Hydrogen peroxide (H2O2) is a reactive oxygen species that also serves as an important signaling molecule in normal brain function. Because H2O2 serves these distinct biological roles, H2O2 concentrations likely rise and fall in the extracellular space with precise spatial and temporal resolution, such that functional levels can be achieved for signaling while the pathological consequences resulting from unregulated generation are prevented. However, studies aimed at elucidating these dynamics have been hindered by the lack of a method for probing dynamic H2O2 fluctuations in living systems with molecular specificity. The goals of this research proposal are to enable the quantitative analysis of endogenous H2O2 fluctuations in real-time, and to elucidate how these molecular dynamics modulate those of dopamine (DA) in intact, functional brain tissue. H2O2 is implicated in the pathogenesis of Parkinson's disease. Simultaneous H2O2 and DA measurements will enable regulatory kinetics and mechanisms to be unraveled, investigation of the alteration of these mechanisms by disease or pharmacological agents, and clarification of the neurochemical processes that underlie motor dysfunction. Carbon-fiber microelectrodes will be employed with fast-scan cyclic voltammetry, as this approach provides a quantitative view of neurotransmission in discrete brain locations in real-time. The specific aims combine the development of new technology with innovative applications. They are: 1. To enable the precise characterization of H2O2 fluctuations in the extracellular space of specific brain nuclei, shedding light on its modulatory signaling role, extrasynaptic lifetime, sphere of influence, and diffusion profile under both normal and pathological conditions. These experiments will also demonstrate the extent to which various sources of H2O2 contribute to signaling within select brain nuclei. 2. To elucidate the precise physiological interaction between H2O2 and DA, and the role that these molecular dynamics play in the onset of motor complications associated with Parkinson's disease. In order to achieve these aims, powerful mathematical models will be developed and validated that can be used to interpret the effects of pharmacological agents on the balance between H2O2 generation and clearance. Existing analytical techniques will be modified to enable improved quantitative assessment in the face of chemical variability. The proposed research is significant because the results are expected to vertically advance and expand our understanding of the physiological roles played by H2O2 in the brain, and to shed light on whether oxidative stress is an initiator of dopaminergic dysfunction, or a consequence of that process. Ultimately, such knowledge will inform the development of improved therapeutic interventions, neuroprotective strategies, and promising antiparkinsonian drugs based on redox biology. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because characterization of H2O2 fluctuations in the brain, and elucidation of the precise physiological interaction between H2O2 and dopamine, is expected to help establish the role that these molecular dynamics play in the onset of motor complications associated with Parkinson's disease. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will help to improve health and reduce the burdens of illness.