The long term goal of this research is to establish the chain of molecular events associated with neurotransmitter release at the single cell and subcellular level. This goal includes the characterization of the spatial, and domain structure of phospholipid membrane layers and the temporal behavior of small molecules such as dopamine, seratonin, and histamine involved in the process of exocytosis. The central approach will be to utilize an advanced bioanalytical mass spectrometry-based protocol to acquire two and three dimensional molecule-specific image information at the nanoscale level. The instrumentation involves using a specialized freeze-fracture device which allows cells to be quenched in the laboratory and sectioned in a sample preparation chamber of the mass spectrometer. Mass spectra are acquired by utilizing an energetic beam comprised of molecular cluster ions focused to a sub-micron spot diameter directly onto single cells. Molecular ions are desorbed into a time-of flight mass spectrometer with images constructed by rastering the ion beam over the field of view and collecting mass spectra at each pixel. There are four specific aims for this proposal. First, although adequate sensitivity is available to acquire the requisite molecule-specific images, there are opportunities to build on the capabilities of the instrumentation even further, given recent success in implementation of Au3, Bi3 and C60 cluster beams. Plans include enhancement of secondary ion yields by optimizing the special properties of cluster beams to produce protons in the near surface region of the specimen, molecular depth profiling, possible for the first time with cluster beams where chemical damage buildup is mitigated, 3- dimensional measurements whereby the number of available molecules for imaging is significantly increased, laser post- ionization to detect the desorbed neutral molecules, and implementation of new tandem MS instrumentation that more effectively optimizes the properties of the new cluster sources. Second, to provide a basis for cell imaging experiments, there are plans to expand the repertoire of model membrane systems necessary to establish the efficacy of the mass spectrometry experiments. These model systems include Langmuir-Blodgett films-and liposome spheres doped with varying lipid/protein/small-molecule combinations that can be enticed to form domains and to act as models for artificial exocytosis. Third, these protocols will be utilized to study the dynamics of membrane chemistry and neurotransmission in single cells. Candidates include the study of histamine release from mast cells, the study of membrane chemistry after vesicle fusion and the assay of neurotransmitter levels in the solution (halo) around dense core vs. the core of individual vesicles. These experiments require measurements at single cells, and off single events or vesicles to test the hypotheses put forth. Fourth, a new instrument utilizing an orthogonal TOF geometry with tandem MS will be employed for SIMS imaging at higher mass resolution, and better sensitivity to events that are occurring near the synaptic membrane. This scientific agenda will provide valuable information toward understanding the molecular basis of brain-related disease states such as Alzheimer's and Parkinson's disease and a variety of autoimmune conditions recently hypothesized to involve lipid domains.
We propose to continue to develop an exciting area of mass spectrometric imaging and to apply this method to understand the structure and biophysics of cell membranes in regulating neurotransmitter exocytosis. In addition, cholesterol will be probed as a marker of lipid domains that might play a role in determining, for example, Alzheimer's and autoimmune disease states. A major objective is to establish the unique imaging strategy as a valuable new tool for use by the larger biological and public health research community.
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