Neurophysiological experiments will define basic molecular processes involved in pain and demonstrate molecular interactions occurring during pain perception. We will identify quantity, structure, and metabolites of specific molecular species participating in pain. Experimentally we will focus on a specific function of the fifth cranial nerve system. The tooth pulp-semilunar ganglion-brain stem axis will be the focus of this grant. Opioid peptides will be quantified in these regions and a comparison made between stimulated (chemical, electrical) and unstimulated pulpal tissue. An HPLC-derived metabolic profile of peptides will be obtained. The hypothesis to be tested is that biologically active peptides participating in pain mechanisms will change levels between normal and stimulated states. We will quantify the opioid pentapeptides-leucine enkephalin and methionine enkephalin, dynorphin, beta-endorphin, associated precursors, pentapeptide metabolites, substance P, bradykinin, and PGE2. To achieve quantification of these compounds at endogenous neurophysiological levels, we need to further increase our analytical and molecular sensitivity of quantification to the subnanogram g-I wet weight tissue level. Computer techniques will be developed to average and accumulate multiple alternating linked-scan data to improve the signal-to-noise ratio and increase sensitivity. Furthermore, we will use field desorption and fast atom bombardment mass spectrometry, in combination with collision activation and linked-scanning techniques, to attach absolute molecular specificity to the analysis of protonated molecular ions produced of the peptide(s) of interest. The significance of this grant application includes the achieving of maximum molecular specificity that can be attached to a highly sensitive measurement of endogenous peptides in biologic tissue. Knowledge of the pain-modulating system will explain current pain therapies and create new approaches to treatment of pain. Appropriate and knowledgable stimulation of pain-inhibitory systems could circumvent problems of tissue destruction and addiction. All of these studies will have vast impact on neuroendocrinology, neuropathology, gastroenterology, medical, dental, social, and economic fields.