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来探索活体中的脑化学动力学。大脑中的神经元和神经胶质细胞通过释放神经递质与邻近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。在体内监测化学信息的一种通用和有效的方法是将采样方法(如微渗析)与分析测量相结合。虽然这种方法已经被证明是非常有价值的，但它的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适用于急性测量的限制。在这个项目中，我们将开发技术和方法来解决这些问题。时间分辨率很重要，因为在行为和实验动作过程中，发射器的浓度可能会迅速变化。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色散的限制。我们将开发一种微流控采样系统，将水样流分成油流中的液滴，然后用基于芯片的快速电泳法对液滴进行分析。样本流分割将防止质量传输过程中的分散，并允许许多神经递质的时间分辨率达到10 S或更高。该系统将与微型采样探头相结合，以提高空间分辨率，并允许访问较小的大脑区域。神经肽对多种脑功能有调节作用，但由于目前方法的敏感性，体内监测受到限制，样品采集时间长达30分钟，时间分辨率较低。我们将开发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分析方法。该检测方法对1？L样本的检测下限为下午1：00，从而使神经肽监测的时间分辨率空前提高了10倍。体内的化学测量几乎总是被敏锐地进行；然而，能够在几周内监测神经化学，以监测与疾病相关的进行性变化，如成瘾，或正常功能，如学习，将是极其有用的。长期监测通常被反应性胶质增生所阻止，这是一种组织反应，导致探针被包裹，并阻止从活跃的神经组织中采样。我们将探索利用已知的抑制反应性胶质增生和支持神经再生的化合物进行药物干预，以延长体内监测。最后，我们将进行基础神经科学研究，作为测试方法并向更广泛的神经科学界展示其实用性的一种手段。这些应用包括：1)瘦素受体在调节多巴胺和摄食行为中的作用；2)精神刺激剂对阿片肽的影响；以及3)不同行为表型的神经化学差异，这些差异是易受药物成瘾影响的模型。 精神疾病和神经疾病构成了现代社会中一些最具破坏性和治疗费用最高的疾病。确定这种疾病背后的神经化学失衡是开发适当治疗方法的关键一步；然而，在大多数情况下，神经化学还没有被很好地理解。在这个项目中，我们正在开发新的仪器和技术，使人们能够监测活着的大脑中的神经化学物质。这些新方法将使人们能够解决与大脑疾病的根本原因有关的重要问题，这些疾病涉及成瘾、亨廷顿氏病和肥胖症等各种疾病。

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.