In vivo chemical monitoring using capillary separations

我们的总体目标是开发新的生物分析方法来探索脑化学动力学 活着。大脑中的神经元和神经胶质细胞通过释放神经递质与受体相互作用来进行沟通 相邻的细胞。监测体内神经化学物质和代谢物的浓度动态是至关重要的 在努力了解大脑功能、疾病和治疗方面的工具。一种多用途且有效的方法 化学信息的体内监测是将采样方法(如微渗析)与分析方法相结合 测量。尽管这种方法已被证明是无价的，但其实用性受到时间分辨率较差的限制， 空间分辨率差，神经肽监测结果差，仅适用于急性测量。 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，因为发射器的浓度在 行为和实验动作。时间分辨率通常受到浓度分散的限制 当脉冲被传输到分析系统时。我们将开发一种微流控采样系统 由此水样流被分割成油流中的液滴，并且液滴 随后采用芯片快速电泳法进行分析。样本流分割将阻止 对于许多神经递质，其时间分辨率可达10 S或更高。 该系统将与微型采样探头相耦合，以提高空间分辨率并允许访问 大脑的小区域。神经肽调节许多大脑功能；然而，在体内监测它们是有限的。 由于目前方法的敏感性，使得样品采集必须在~30分钟内进行，从而导致时间不佳 决议。我们将开发基于毛细管液相色谱和 微流控免疫分析。对于1微米L样品，检测下限为1 PM，允许 神经肽监测的时间分辨率史无前例地提高了10倍。体内化学 测量几乎总是被敏锐地执行；然而，如果能够 在一段时间内监测神经化学，以监测与疾病相关的进行性变化，如 上瘾，或正常功能，如学习。长期监测通常由反应性胶质细胞增多症，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取样。我们 将探索使用已知的抑制反应性胶质细胞增生的化合物的药物干预和 支持神经再生，延长体内监测时间。最后，我们将进行基础神经科学 研究作为测试这些方法并展示它们对更广泛的神经科学的效用的一种手段 社区。这些应用包括确定：1)瘦素受体在调节多巴胺和 摄食行为；2)精神刺激剂对阿片肽的影响；3)神经化学差异 潜在的不同的行为表型是易受药物成瘾影响的模型。

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 µL 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.