The BRAIN Initiative seeks to understand the spatial, temporal and chemical nature of the brain. RFA-MH-15- 225 calls for the development of new tools and technologies with a number of goals, including methods to obtain cell type and chemical information from individual cells and their connections. While many imaging approaches exist that use specific probes to image subsets of cells and their interconnections, this project will create a chemical information-rich approach that advances the emerging technique of stimulated Raman scattering microscopy (SRSM). SRSM provides vibrational spectral data from every location of a living brain slice so that dynamic chemical changes can be followed. The Raman spectra contain tremendous chemical information but the data is coded in complex overlapping molecular vibrational bands. With appropriate training sets-derived from the Raman data and comparing it to the chemical contents of individual cells-a series of mathematical models will be developed that create unlimited Computational Histology maps. In order to (a) inform the mathematical model in the development phase and (b) greatly augment the chemical information obtained from these studies, dissociated cells will be subjected to another measurement-high throughput single cell mass spectrometry (MS)-on tens of thousands of cells. Single cell MS provides detail on hundreds of components in each cell, effectively mapping each cells' peptidome and metabolome. The MS data includes unique information on the metabolic state of these cells and allows us to define known and unknown cell types. Computational models will be used to correlate the SRSM data to the MS-derived chemical content as well as deliver strategies to examine the dynamic changes and heterogeneity in brain tissue. These technologies will be validated using the dentate gyrus. The focus of the work will be on the hippocampal neurons and glia of the dentate gyrus and their involvement in memory formation, and issues related to astrocyte morphology changes. By performing patch clamp physiological measurements and detailed MS-based metabolomic profiling on the patched cells of the dentate gyrus, the SRSM and single cell MS technology platform will be validated by investigating this complex area of the brain containing many cell types, heterogeneous morphologies, and chemical characteristics. These technologies will provide unmatched detail on the chemical content and dynamics within this defined brain region, answer long intractable questions related to cellular heterogeneity, and relate this information to organization and functional processes such as long term potentiation.
A novel analytical platform, integrating the strengths of mass spectrometry and stimulated Raman scattering microscopy, will be developed to characterize brain cells and tissues with unmatched chemical detail. This technology platform directly addresses an unmet need for the BRAIN initiative, will lead to new neuroscience insights, and help create novel diagnostic and therapeutic opportunities.
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