New Technology for In Vivo Monitoring the Brain Extracellular Proteome at High Spatial Resolution in Substance Abuse Models

项目摘要。 这个项目的总体目标是开发新的方法来采样、识别和量化 分泌到脑细胞外间隙的蛋白质与物质使用障碍(SUD)有关。这个 发展为强迫性药物消费与神经元结构和 神经递质对药物的反应释放。有证据表明，细胞外空间中的蛋白质在 在这些变化中扮演了一个角色。例如，脑源性神经营养因子(BDNF)释放到细胞外 空间可能会刺激与药物滥用适应相关的神经元变化；然而， 实际上，还没有对大脑细胞外空间中的蛋白质进行直接测量。跟踪的能力 体内某些神经递质的释放在过去20年里取得了巨大的进步，并导致了 我们对它们的释放如何被SUD改变和促进的理解有了深刻的提高。打开一个 了解蛋白质如何动态变化的窗口将提供更多的见解。一种强大的方法来 监测是与分析技术相结合的活体采样。这种方法，以微透析为例 采样在揭示大脑中多巴胺和其他小分子的动态方面发挥了重要作用 功能和药物滥用。这种方法在追踪蛋白质方面不太成功，因为 这些分子的回收。低回收率是由于蛋白质向采样设备的低扩散以及 对透析膜的吸附。这样的探头也太大了，无法对许多大脑核团进行采样。在这里我们 描述新的采样系统，它将在1000的空间分辨率下对蛋白质有更高的回收率- 折叠效果比微透析好。我们将开发一种新的采样方法，这种方法可以诱导电驱动流 通过大脑将蛋白质送入采样毛细管。这种电渗透推挽灌流(EOPPP) 探测器将接受测试，以确定其回收蛋白质的能力。这种方法的有效性将与 另一种基于低流量推拉灌流的新探头。在这种方法中，一个微型制造的探头，几十个 宽度和厚度均为微米，只有一个通道可以取出样品，第二个通道可以输送样品 置换液速度为~100 NL/分钟。 在测试和开发这些采样探针之后，我们将开发蛋白质组学方法来 分析胞外蛋白质组。这些方法将基于微量样品的制备来消化 从蛋白质到组成多肽、先进的多肽高分辨率分离以及最新技术 利用质谱学和信息学进行蛋白质组学分析。我们将鉴定细胞核中的胞外蛋白 伏隔核和前额叶皮质是第一次。然后，我们将比较对照组的细胞外蛋白质组 与在经过自我注射可卡因的动物身上发现的结果相同，可卡因是一种经过验证的苏丹明模型。这项工作将揭示 新的SUD神经底物，并为在SUD模型中进一步探索脑蛋白质组奠定了基础。 。

Project Summary. The overall objective of this project is to develop novel approaches to sample, identify, and quantify proteins secreted into the brain extracellular space in relation to substance use disorders (SUD). The progression to compulsive drug consumption is associated with changes in neuronal structure and neurotransmitter release in response to drugs. Evidence suggests that proteins in the extracellular space play a role in these changes. For example, brain derived neurotrophic factor (BDNF) released to the extracellular space may stimulate neuronal changes that are associated with adaptation to drugs of abuse; however, practically no direct measures of proteins in the brain extracellular space have been made. The ability to track release of certain neurotransmitters in vivo has dramatically advanced over the past 2 decades and led to profound increases in our understanding of how their release is altered by and contributes to SUD. Opening a window onto how proteins dynamically change will provide even greater insights. A powerful approach to monitoring is in vivo sampling coupled to analytical techniques. This approach, exemplified by microdialysis sampling, has been instrumental in uncovering dynamics of dopamine and other small molecules in brain function and substance abuse. This method has been less successful for tracking proteins due to low recoveries of these molecules. Low recovery is due to low diffusion of proteins to the sampling device and adsorption to dialysis membranes. Such probes are also too large to sample many brain nuclei. Here we describe novel sampling systems that will have higher recovery for proteins at spatial resolution that is 1000- fold better than microdialysis. We will develop a new sampling method that induces electrically driven flow through the brain to drive proteins into a sampling capillary. This electroosmotic push-pull perfusion (EOPPP) probe will be tested for its ability to recover proteins. The effectiveness of this method will be compared to another new probe based on low-flow push-pull perfusion. In this method, a microfabricated probe, tens of micrometers in width and thickness, has a single channel to withdraw sample and a second to deliver replacement fluid at ~100 nL/min. After testing and development of these sampling probes, we will then develop proteomic methods to analyze the extracellular proteome. These methods will be based on microscale sample preparation to digest proteins to component peptides, advanced high-resolution separation of peptides, and state-of-the art proteomic analysis by mass spectrometry and informatics. We will identify extracellular proteins in the nucleus accumbens and prefrontal cortex for the first time. We will then compare the extracellular proteome in controls to that found in animals trained to self-administer cocaine, a validated model of SUD. This work will uncover new neural substrates of SUD and set the stage for further exploration of the brain proteome in SUD models. .