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 分泌到脑细胞外空间的蛋白质与物质使用障碍（SUD）有关。的 强迫性药物消耗的进展与神经元结构的变化有关， 神经递质的释放。有证据表明细胞外空间的蛋白质 在这些变化中的作用。例如，脑源性神经营养因子（BDNF）释放到细胞外， 空间可刺激与适应滥用药物相关的神经元变化;然而， 实际上还没有对脑细胞外空间中的蛋白质进行直接测量。能够跟踪 体内某些神经递质的释放在过去的20年中已经有了显著的进展， 深刻增加了我们对它们的释放如何被SUD改变和促成SUD的理解。打开 了解蛋白质如何动态变化的窗口将提供更深入的见解。一种强大的方法， 监测是结合分析技术的体内取样。以微透析为例， 采样，一直有助于揭示多巴胺和其他小分子在大脑中的动力学 功能和药物滥用。这种方法在跟踪蛋白质方面不太成功，因为低的 回收这些分子。低回收率是由于蛋白质向采样装置的低扩散， 吸附到透析膜上。这种探针也太大，无法对许多脑核进行采样。这里我们 描述了新颖的采样系统，其将在1000- 2000的空间分辨率下具有更高的蛋白质回收率。 比微透析好一倍。我们将开发一种新的采样方法， 通过大脑将蛋白质送入取样毛细管。电渗推拉灌注（EOPPP） 将测试探针回收蛋白质的能力。该方法的有效性将与 另一种基于低流量推挽灌注的新探头。在这种方法中，微加工探针，数十个 宽度和厚度均为20微米，具有单个通道用于提取样品，第二个通道用于输送样品。 置换液流速约为100 nL/min。 在测试和开发这些取样探针之后，我们将开发蛋白质组学方法， 分析细胞外蛋白质组这些方法将基于微量样品制备来消化 蛋白质到组分肽，肽的先进高分辨率分离，以及最新技术水平 通过质谱和信息学进行蛋白质组学分析。我们将鉴定细胞核中的细胞外蛋白质 大脑皮层和前额叶皮层。然后，我们将比较对照组的细胞外蛋白质组， 在训练自我施用可卡因的动物中发现的，这是一种经过验证的SUD模型。这项工作将揭示 新的神经底物的SUD和设置阶段，进一步探索脑蛋白质组的SUD模型。 .

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. .