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着根本的差距。因此，长期目标是阐明大脑中几个活性氧物种和小分子的释放/清除动力学是如何导致涉及氧化应激的神经退行性疾病状态的。过氧化氢(H_2O_2)是一种活性氧物种，也是正常脑功能的重要信号分子。由于H_2O_2具有这些不同的生物学作用，H_2O_2浓度可能在细胞外空间以精确的空间和时间分辨率上升和下降，这样就可以达到信号的功能水平，同时防止由于不受调控的产生而导致的病理后果。然而，由于缺乏一种具有分子特异性的方法来探测生命系统中的动态H_2O_2波动，旨在阐明这些动力学的研究一直受到阻碍。这项研究计划的目标是能够实时定量分析内源性过氧化氢的波动，并阐明这些分子动力学如何调节完整、功能正常的脑组织中的多巴胺(DA)的波动。过氧化氢与帕金森氏病的发病机制有关。H_2O_2和DA的同步测量将有助于解开调节动力学和机制，研究疾病或药物对这些机制的改变，并澄清导致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，因为这种方法实时提供了离散大脑位置的神经传递的定量视图。具体目标是将新技术的开发与创新应用相结合。它们是：1.为了能够准确地描述特定脑核细胞外间隙中H_2O_2的波动，揭示其在正常和病理条件下的调制信号作用、突触外寿命、影响范围和扩散情况。这些实验还将展示不同来源的过氧化氢在选定脑核内对信号的贡献程度。2.阐明H_2O_2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的发生中所起的作用。为了实现这些目标，将开发和验证强大的数学模型，这些模型可以用来解释药物对过氧化氢产生和清除之间平衡的影响。将对现有的分析技术进行修改，以便在面对化学变异性时能够改进定量评估。这项拟议的研究意义重大，因为这些结果有望垂直推进和扩大我们对过氧化氢在大脑中扮演的生理角色的理解，并阐明氧化应激是多巴胺能功能障碍的始发者，还是这一过程的结果。最终，这些知识将为改进的治疗干预措施、神经保护策略和基于氧化还原生物学的有前景的抗帕金森药物的开发提供信息。 公共卫生相关性：这项拟议的研究与公共健康相关，因为对大脑中过氧化氢波动的表征，以及对过氧化氢和多巴胺之间精确的生理相互作用的阐明，有望有助于确定这些分子动力学在与帕金森氏病相关的运动并发症的发生中所起的作用。因此，拟议的研究与NIH使命的一部分有关，该部分涉及发展有助于改善健康和减轻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.