Analgesic tolerance arises with chronic opioid pharmacotherapy. Blockade of NMDA receptors reduces the development of opioid tolerance, but delivery of NMDA receptor antagonists can result in motor and/or cognitive toxicity. Agmatine is a decarboxylated form of L-arginine produced in the central nervous system (CNS) that significantly reduces opioid analgesic tolerance. Although evidence suggests that agmatine antagonizes the NMDA receptor, unlike many synthetic NMDA receptor antagonists delivered spinally, it does not elicit motor deficits. This pharmacological profile suggests that spinally delivered agmatine may act at the NR2B subunit of the NMDA receptor, the expression of which is restricted to the sensory region of the spinal cord and not in the ventral horn. The objective o this application is to define the mechanism by which CNS agmatine modifies behavioral neuroplasticity, evaluate its role as a neuromodulator, and determine the potential of targeting the agmatinergic system as a gene therapy to improve opioid analgesia. The central hypothesis is that CNS agmatine inhibits opioid analgesic tolerance through antagonism of the NMDA receptor and in particular the NR2B-containing NMDA receptors. Three specific aims are proposed: Specific Aim #1: Delineate the contribution of CNS-derived agmatine to inhibition of opioid tolerance. We will address this aim through evaluation of induction of spinal opioid tolerance under conditions of genetic alteration of agmatine's synthetic and degradative enzymes in vivo. Specific Aim #2: Define the mechanism by which CNS-derived agmatine inhibits opioid tolerance. We will address this aim with pharmacological, physiological, and molecular approaches that will test the relationship of agmatine to the glutamatergic system. Specific Aim #3: Determine the impact of opioid tolerance on the agmatinergic system and its target receptor(s). To address this aim, we will use anatomical and molecular approaches. The experimental approach is innovative because it will uncover a largely understudied inhibitory system that opposes maladaptive neuroplasticity. The information acquired will indicate the extent to which endogenous agmatine can be accessed to maximize inhibition of pathological neuroadaptation (e.g. opioid tolerance). The knowledge gained is expected to be useful for management and treatment of chronic pain and directly applicable to other CNS dysfunctions.