Understanding brain function in healthy and diseased brains requires an understanding of the biochemistry occurring within the neurons and supporting cells making up the brain. It is well known that individual cells in the brain have distinct signaling molecule complements and protein complement, but the effects on the cellular metabolome are much less well understood. What is the cell to cell variation of the cellular metabolome in the mammalian CNS? For most metabolites, the answer is unknown. Using invertebrate models, large differences in the metabolome of adjacent neurons have been measured such as millimolar levels of nitrate in nitrergic neurons with no detectable nitrate in non-NO producing neurons. Similarly, large changes in the amino acid complement occur depending on the signaling molecules used by the neuron. Unfortunately, technology limitations do not currently allow the major metabolites to be measured within individual mammalian neurons. A suite of technology development and hyphenated approaches are proposed to create instruments and protocols to measure the metabolites in neuronal clusters, groups of neurons and even individual neurons. These development efforts include unique sampling protocols, microfluidically-based sample conditioning unit with integrated electrophoretic separations, followed by native fluorescence and mass spectrometric detection, and where necessary, capture of the appropriate metabolites into nanolitervolume capillaries for nanoliter volume NMR spectroscopic characterization. This unique set of technology promises to open up a new volume regime to profile the cell to cell variations in the metabolites. The technology will be tailored to and validated on the most heterogeneous samples known - the mammalian brain.
