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

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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.