In vivo chemical monitoring using capillary separations

描述（由申请人提供）：我们的总体目标是开发新的生物分析方法，用于探索体内脑化学动力学。大脑中的神经元和神经胶质细胞通过释放神经递质与相邻细胞上的受体相互作用来进行交流。监测体内神经化学物质和代谢物的浓度动态是了解大脑功能、疾病和治疗的重要工具。一个通用的和有效的方法，在体内监测的化学信息是耦合采样方法，如微透析，分析测量。虽然这种方法已被证明是非常宝贵的，但其实用性受到时间分辨率差、空间分辨率差、神经肽监测结果差以及仅应用于急性测量的限制。在本项目中，我们将开发解决这些问题的技术和方法。时间分辨率是重要的，因为发射器的浓度可以在行为和实验操作期间迅速变化。当浓度脉冲被传输到分析系统时，时间分辨率通常受到浓度脉冲的分散的限制。我们将开发一种微流控采样系统，其中含水样品流被分割成油流内的液滴，随后通过基于芯片的快速电泳分析液滴。样本流分割将防止在质量运输过程中的分散，并允许10秒或更好的时间分辨率为许多神经递质。该系统将与小型化的采样探针相结合，以提高空间分辨率，并允许进入小的大脑区域。神经肽调节许多脑功能;然而，在体内监测它们受到当前方法的灵敏度的限制，使得样品必须收集约30分钟，导致时间分辨率差。我们将发展基于毛细管液相色谱和微流控免疫分析的高灵敏度神经肽检测方法。该测定将有1 pM的检测限为1？L样品，使神经肽监测的时间分辨率前所未有的10倍提高。体内化学测量几乎总是急性进行的;然而，能够在数周内监测神经化学，以监测与疾病（如成瘾）或正常功能（如学习）相关的进展性变化，将是非常有用的。长期监测通常会因反应性神经胶质增生而受到阻碍，这是一种导致探针包裹并阻止从活性神经组织中采样的组织反应。我们将探索使用已知的抑制反应性神经胶质增生和支持神经再生的化合物的药理学干预，以延长体内监测。最后，我们将进行基础神经科学研究，作为测试方法的一种手段，并向更广泛的神经科学界展示其实用性。这些应用包括确定：1）瘦素受体在调节多巴胺和进食行为中的作用; 2）精神兴奋剂对阿片肽的影响; 3）作为药物成瘾易感性模型的不同行为表型的神经化学差异。 精神疾病和神经系统疾病是现代社会中最具破坏性和最昂贵的治疗疾病。确定这些疾病背后的神经化学失衡是开发适当疗法的关键步骤;然而，在大多数情况下，神经化学并没有得到很好的理解。在这个项目中，我们正在开发新的仪器和技术，使神经化学物质在活的大脑中被监测。这些新方法将使一些重要的问题得以解决，这些问题涉及成瘾、亨廷顿病和肥胖等多种大脑疾病的根本原因。

DESCRIPTION (provided by applicant): Our overall objective is to develop new bioanalytical methods for exploring brain chemistry dynamics in vivo. Neurons and glia in the brain communicate by releasing neurotransmitters that interact with receptors on neighboring cells. Monitoring the concentration dynamics of neurochemicals and metabolites in vivo is a vital tool in the effort to understand brain function, diseases, and treatments. A versatile and effective approach for in vivo monitoring of chemical messages is to couple sampling methods, such as microdialysis, to analytical measurements. Although this approach has proven invaluable, its utility is limited by poor temporal resolution, poor spatial resolution, poor results for neuropeptide monitoring, and application to only acute measurements. In this project, we will develop technology and methods to solve these problems. Temporal resolution is important because concentrations of transmitters can change rapidly during behavior and experimental maneuvers. Temporal resolution is often limited by dispersion of concentration pulses as they are transported to the analytical system. We will develop a microfluidic sampling system whereby the aqueous sample stream is segmented into droplets within a stream of oil and the droplets subsequently analyzed by rapid chip-based electrophoresis assays. Sample stream segmentation will prevent dispersion during mass transport and allow temporal resolution of 10 s or better for many neurotransmitters. This system will be coupled to miniaturized sampling probes to improve spatial resolution and allow access to small brain regions. Neuropeptides regulate many brain functions; however, monitoring them in vivo is limited by the sensitivity of current methods so that samples must be collected for ~30 min resulting in poor temporal resolution. We will develop high sensitivity neuropeptide assays based on capillary liquid chromatography and microfluidic immunoassays. The assays will have detection limits of 1 pM for 1 ?L samples allowing an unprecedented 10-fold improvement in temporal resolution for neuropeptide monitoring. In vivo chemical measurements are nearly always performed acutely; however, it would be extremely useful to be able to monitor neurochemistry over a period of weeks to monitor progressive changes associated with diseases, like addiction, or normal function, like learning. Long term monitoring is typically prevented by reactive gliosis, a tissue reaction that results in encapsulation of the probe and prevents sampling from active neural tissue. We will explore the use of pharmacological interventions with compounds known to suppress reactive gliosis and support neuroregeneration to prolong in vivo monitoring. Finally, we will perform fundamental neuroscience studies as a means of testing the methods and demonstrating their utility to the broader neuroscience community. These applications include determining: 1) the role of leptin receptors in regulating dopamine and feeding behavior; 2) the effect of psychostimulants on opioid peptides, and 3) neurochemical differences underlying distinct behavioral phenotypes that are a model for vulnerability to drug addiction. Mental illnesses and neurological diseases comprise some of the most devastating and expensive to treat disorders in modern society. Determining the neurochemical imbalances underlying such disorders is a key step in developing appropriate therapies; however, in most cases the neurochemistry is not well understood. In this project, we are developing novel instrumentation and techniques that enable neurochemicals to be monitored in the living brain. These new methods will enable important questions to be addressed relating to underlying causes of diseases involving the brain as diverse as addiction, Huntington's disease, and obesity.