Chronic pain is an important and largely unresolved medical problem. Substance P and other neurokinins (NKs), acting on the neurokinin 1 receptor (NK1R), produce changes in dorsal horn neurons of the spinal cord related to the onset of chronic pain. Conversely, opioid peptides, acting on the mu-opioid receptor (MOR), may alter dorsal horn neurons to relieve chronic pain. The overall goal of this proposal is to understand mechanisms that control the release of NKs and endogenous opioid peptides in the dorsal horn. The project will test the following hypotheses: 1) High frequency firing of A-fibers facilitate NK release from C-fiber terminals. 2) This facilitation by A-fibers is mediated by two inhibitory interneurons: one producing sustained inhibition of NK release through presynaptic GABAB receptors, another inhibiting the first through GABAA receptors. 3) In the dorsal horn, opioid peptides are released by primary afferents firing at low frequency, and also by interneurons receiving adrenergic signals. The specific aims are to 1) determine the role of firing frequency and of A-fibers in controlling NK release from C-fibers; 2) characterize the control of NK release by GABA receptors; 3) determine the role of primary afferents and adrenergic receptors in the release of opioid peptides. NK1Rs and MORs get internalized by the neurons after their activation by agonists. Hence, NK1R and MOR internalization, detected by immunofluorescence staining, will be used to measure the release of NKs and opioids, respectively. This approach has the advantages of determining release in terms of receptor activation, identifying the area of diffusion of the peptide and the morphology of the target neurons, and detecting the release of any endogenous agonist able to internalize the receptor. To confirm that receptor internalization correlates with neuropeptide release, release will be measured also by radioimmunoassay (RIA), and the depletion of neuropeptides in the dorsal horn will be assessed by immunohistochemistry. This project pioneers the combination of these techniques with the use of spinal cord slices with attached dorsal roots. Slices have the advantage of allowing the controlled delivery of drugs in combination with well-defined patterns of electrical stimulation. Once experimental conditions are refined in slices, they will be applied to studies ''n vivo'' to further test the hypotheses in the whole animal.