Specific Aims: Our specific biological aim is to characterize the importance of neuropeptides in stroke-like conditions. We hypothesize that low levels of free radicals may actually stimulate the synthesis and release of neuropeptides that protect against harm from higher levels of free radicals. We hypothesize that high levels of free radicals cause the release of toxic neuropeptides and/or decrease the synthesis and release of protective peptides. We will target several specific peptides including dynorphins, enkephalins, substance P, and neuropeptide Y. Our technical aims are to develop trace, quantitative analysis of peptides and their inactive fragments. A new, noninvasive sampling procedure for use in brain slices will be developed based on a sound theoretical framework. This sampling technique will realize high yields of low and high molecular weight peptides continuously over time while interfaced to high performance liquid chromatography (HPLC), capillary electrophoresis, electrospray mass spectrometry and related techniques. Lower detection limits and a more robust system will result from novel electrochemical detectors and a newly developed fluorescence scheme. Health Relatedness. Neurotrophic and protective peptides help the brain to respond to challenges, either intellectual or pathological. Pharmacological stimulation of the synthesis of these peptides would help the brain to protect and heal itself. Our methodological advances will accelerate understanding of neuroprotective peptides, and may be applied to a wide range of other peptide-related questions. Methods. The sampling technique will be modeled theoretically to understand the interplay among factors that control the recovery of analytes from brain slices. Model slices will be used to confirm theoretical predictions. A total analytical system will be created based on the novel sampling technique, an interface to HPLC, and an electrochemical detection system. Incremental advances in technology will be made to a working system to improve separation capability, robusmess, reproducibility and accuracy. Organotypic hippocampus slices will be challenged with subtoxic and toxic levels of glutamate agonists and free radical generators. Peptide release will be determined in parallel with other measures of slice activity and viability.
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