Deep Analysis of Brain Chemistry at Enhanced Spatial and Temporal Resolution using Microscale Sampling and Analysis

本计画的目的是发展新的生物分析方法，以探索活体脑化学动力学。监测体内神经化学物质的浓度动态对于研究大脑功能、疾病和治疗至关重要。一种用于脑化学的体内监测的通用方法是将采样方法（例如微透析）与分析测量相结合。相对于其他工具（如传感器），采样方法的主要优点是能够随时间进行连续测量，测量各种化学品，以及代谢追踪的潜力;然而，这些潜在的优势尚未开发。该方法的缺点是时间和空间分辨率差。时间分辨率受到收集足够样本进行分析所需时间的限制，而空间分辨率受到探针大小的限制。在这个项目中，我们将开发技术来克服这些限制。此外，将发展优势和独特的能力，以满足人才倡议的需要。 为了便于在高空间分辨率下进行长期测量，将开发新型微加工采样探针，以允许在几个细胞的尺度上进行测量。为了提高时间分辨率，将开发用于将基于质谱法和邻近免疫测定法的快速测定与新的采样探针相结合的仪器。这些方法将允许连续测量神经递质，代谢物和蛋白质的实时1秒的时间分辨率。第二个目标是开发代谢组学，即测量从体内脑细胞外空间收集的样本中存在的脂质和代谢物的全部补充。为此，将开发超高分辨率毛细管液相色谱-质谱联用技术，以分析探针样品中存在的代谢物组。这种方法将允许大脑细胞外空间的化学环境随着时间的推移和在特定的大脑位置以前所未有的细节来表征。通过这种方式，将有可能发现控制不同精神和疾病状态的化学物质和代谢途径。第三个目标将是使用稳定同位素示踪来检测谷氨酸和GABA的神经元库，这是大脑中的主要神经递质。这些化学物质具有多种细胞来源，因此先前的研究在理解检测到的浓度变化是否是由于其他细胞活动的神经元功能方面受到限制。这种能力将使人们对这些神经递质的动力学有一个新的理解，这些神经递质参与了无数的大脑功能。

The objective of this project is to develop new bioanalytical methods for exploring brain chemistry dynamics in vivo. Monitoring the concentration dynamics of neurochemicals in vivo is vital for studying brain function, diseases, and treatments. A versatile approach for in vivo monitoring of brain chemistry is to couple sampling methods, such as microdialysis, to analytical measurements. Primary advantages of the sampling methods relative to other tools, such as sensors, are the ability to perform continuous measurements over time, measure a wide variety of chemicals, and potential for metabolic tracing; however, these potential advantages are underdeveloped. The disadvantages of the method are the poor temporal and spatial resolution. Temporal resolution is limited by the time required to collect enough samples for analysis while spatial resolution is limited by the probe size. In this project, we will develop technology to overcome these limitations. Further, the strengths will be developed and the unique capabilities to address the needs of the Brain Initiative. To facilitate long-term measurements at the high spatial resolution, novel microfabricated sampling probes will be developed that allow measurements at the scale of just a few cells. To improve temporal resolution, instrumentation for coupling fast assays based on mass spectrometry and proximity immunoassay to the new sampling probes will be developed. These methods will allow continuous measurement of neurotransmitters, metabolites, and proteins in real-time with 1 s temporal resolution. A second aim is to develop metabolomics, i.e. measurement of the full complement of lipids and metabolites present, for samples collected from the brain extracellular space in vivo. For this aim, ultra-high-resolution capillary liquid chromatography coupled to mass spectrometry will be developed to analyze the metabolome present in probe samples. This method will allow the chemical milieu of the brain extracellular space to be characterized in unprecedented detail over time and at specific brain locations. In this way, it will be possible to discover chemicals and metabolic pathways that govern different mental and disease states. A third aim will be to use stable-isotope tracing to detect the neuronal pool of glutamate and GABA, the primary neurotransmitters in the brain. These chemicals have multiple cellular sources so previous studies have been limited in understanding if detected changes in concentration were due to neuronal function of other cell activities. This capability will allow a new understanding of the dynamics of these neurotransmitters, which are involved in a myriad of brain functions.