CORE--ANALYTIC NEUROCHEMISTRY AND SPECTROSCOPY CORE

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The past two decades have seen enormous growth in our understanding of brain function, especially of those events that form the basis of synaptic transmission. Hundreds of laboratories have labored diligently to characterize the metabolism, pharmacology, transport and development of the neurotransmitters and the synaptic apparatus. The fortunate outcome of this enterprise is a detailed understanding of the intricacies of information exchange in the nervous system. We can identify the molecular basis for fast and slow neural transmission. We appreciate the importance of glial cells in maintaining ionic homesostasis in the neuropil, in terminating neurotransmission and in furnishing to neurons the precursors for replenishment of the neurotransmitter pool. We have begun to appreciate that the synapse - like the brain itself- is not a finished product, but a structure in a state of perpetual evolution, the development of which proceeds according to a carefully wrought schedule that facilitates the acquisition of new information and the adaptation to an ever changing external environment. Scientific interest in synaptic biochemistry reflects both the fundamental importance of the subject and the recognition that neurotransmitter imbalance is at the heart of many human diseases, among them hypoxic brain injury, inborn errors of metabolism, epilepsy, substance abuse, depression, etc. Mental retardation and other developmental disabilities commonly result from derangements of synaptic biochemistry and/or synaptogenesis. Effective treatments for these disorders presuppose an understanding of the relevant pathobiology. We structured the Analytical Neurochemistry and Spectroscopy Core to provide our users with an array of services that accommodates comprehensive scrutiny of the synapse - from the in vitro analysis of individual metabolites to in situ characterization of ionic flux to in vivo measurement of neurotransmitters and neurotransmitter receptors and transporters. We have been able to make available sophisticated technologies - mass spectrometry, positron emitting tomography, optical imaging - that exceed the analytic capability of an individual laboratory. Furthermore, we support our users by familiarizing each of them with our analytical potential and by helping each user to interpret experimental results. At the initial submission of the MRDDRC, this core sponsored three services: liquid chromatographic (HPLC) analysis of neurotransmitters and their metabolites, GC-MS technology, and a neuronal cell culture facility. In 1995 the current Cellular Neuroscience Core (Core A) was "teased" out as a separate core to improve our repertoire of cellular and pathology services. The current Analytical Neurochemistry and Spectroscopy Core (Core D) then assumed its present configuration. We still provide HPLC resources for the quantitation of amino acids and biogenic amines with high sensitivity and we furnish mass spectrometry services for the determination of stable isotope enrichment for kinetic studies, both in vivo and in vitro. In 2000 we added a Cell Based Assay Facility that measures molecules that are activated by cell death and/or apoptosis, including Ca2+, reactive oxygen species, Ca2+-activated proteases, and caspase-3. We also measure DNA fragmentation (a late marker of apoptosis), lactate dehydrogenase (a cytoplasmic marker of cell death) and the formazan 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazoliumbromide (MTT), a mitochondrial marker for living cells. We added a Positron Emission Tomography (PET) Facility to elucidate molecular and cellular synaptic processes in the living organism. A novel initiative is the current proposed Proteomics service that will enable protein identification in complex mixtures as well as the characterization of sites of post-translational modification. The significance of this Core is reflected in the many publications and grant applications that the core services successfully enabled. Several new scientific initiatives were feasible because of the rich analytic infrastructure that the Core affords. One example is the Rare Disease Research Consortium, a venture that studies the urea cycle defects and that represents a notable example of collaboration among several other MRDDRCs1. We anticipate this trend to continue as we add novel services to the Core, including the Proteomics Facility. This Core contains several different analytic components: HPLC, mass spectrometry, cell-based assays, PET and proteomics. We acknowledge that this repertoire is broad, but we wanted to accommodate our users by creating a single unit that facilitates biochemical study along a continuum extending from individual analytes (HPLC, mass spectrometry and proteomics) to intact cells (cell-based assays) to the whole organism (PET). Not only does this arrangement confer organizational unity, it also allows increased economy, a consideration that we must weigh carefully at a time of strict limits on the overall budget of the MRDDRC. The creation of additional cores would have made impossible such economization. Instead, the current structure allows us to leverage institutional support, which comprises by far the greater proportion of the actual costs of providing our users,with these stateof- the-art services.