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. Specifically, the spatial and temporal behavior of small molecules such as dopamine, serotonin, and histamine, and the domain structure of phospholipid membrane bilayers involved in the process of exocytosis will be determined. To establish these structures, a mass spectrometry-based molecule-specific imaging protocol is being developed. This protocol utilizes a specialized freeze-fracture device that 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 a less than 100 nm-focused energetic ion beam to desorb molecular ions into a time-of flight mass spectrometer. Images are constructed by rastering the ion beam over the target cells and collecting mass spectra at each pixel. There are three aims for this proposal. First, although adequate sensitivity is available to acquire the requisite molecule-specific images, there are emerging possibilities that can significantly boost signal levels, and hence lateral resolution. Plans include implementation of gold and C60 ion sources constructed over the last two years, molecular depth profiling in an ice matrix whereby the number of available molecules for imaging is significantly increased, laser postionization to detect the desorbed neutral molecules, and laser desorption imaging directly from ice using femtosecond UV pulses for mapping protein signals. 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. Model systems include Langmuir-Blodgett films doped with cholesterol that form rafts and liposome networks. The networks can be enticed to form domains and 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 the dense core vs. the core of individual vesicles. To test the hypotheses put forth, measurements of vesicles and single events specific to individual cells are required. This scientific agenda will provide valuable information toward understanding the molecular basis of brain-related disease states, which according to recent hypotheses involve lipid rafts. These diseases include Alzheimer's, Parkinson's and a variety of autoimmune conditions.
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