Intraspinal opioids provide excellent analgesia, but are limited by side effects and often fail to relieve neuropathic pain. In contrast to opioids, alpha2-adrenergic agonists do not cause nausea or depress respiration and are effective in patients with neuropathic pain. The focus of this grant remains to understand the mechanism of spinal alpha2- adrenergic agonist-induced analgesia and side effects. We have shown in the last grant cycle that spinal neostigmine enhances alpha2-adrenergic analgesia and counteracts its hypotension; this follows from our finding that spinal acetylcholine (ACh) causes analgesia and increased blood pressure. This has led to clinical development of spinal neostigmine, under separate NIH funding. Our new preliminary data suggest that both beneficial effects of neostigmine (enhanced analgesia; stable blood pressure) are mediated by spinal nitric oxide (NO). This proposal will examine key questions regarding spinal alpha2-ACh-NO interactions with a variety of methods. Do alpha2-agonists act via ACh release in the dorsal horn? (behavioral studies; microdialysis; intracellular recording) How do alpha2-agonists and ACh regulate NO release? Are IV opioids, which activate descending noradrenergic pathways, mediated by ACh and NO release? Do cholinergic neurons synthesize NO which acts in the same cell to stimulate ACh release? (behavioral studies; microdialysis; intracellular recording; bioassay; 14C-Citrulline release). How do alpha2 agonists, ACh and NO interact to regulate preganglionic sympathetic nerve activity? (renal sympathetic nerve recording; microdialysis) Immunostaining for NO synthase, choline acetyltransferase, and alpha2-receptor subtypes will define anatomical substrates underlying these interactions. Spinal injection and behavioral testing are crude measures to examine drug mechanisms and interactions. To overcome these limitations, we propose a new consortium agreement with Dr. P. Max Headley, one of only 2 investigators worldwide currently using an awake animal model for dorsal horn electrophysiologic recording, and the only one to do so using sheep, the species we have used as the best model for human spinal analgesia. These crucial experiments will detail the precise alpha2-ACh-NO interactions hypothesized in normal animals and those with a naturally occurring clinical pain syndrome. Together, these studies will provide fundamental understanding of a novel hypothesis of regulation of sensory transmission in the spinal cord and mechanisms of action of IV opioids.