Preclinical behavioral pharmacological and neuropharmacological evidence indicates that the NMDA receptor plays an important role in opioid dependence, however, the neural substrates subserving these actions are poorly understood. The central nucleus of the amygdala (CeA) is a critical coordinator of autonomic, behavioral, and emotional systems impacted by opioids, however there is no evidence that the essential NMDA-NR1 (NR1) subunit gene in the amygdala plays a role in opioid dependence. To determine the role of the NR1 subunit gene in the amygdala with respect to physical and psychological opioid withdrawal, a spatial-temporal deletion of this gene was produced by microinjecting a recombinant adeno-associated virus (rAAV) expressing the GFP reporter and Cre recombinase (rAAV-GFP-Cre) into the CeA of adult "floxed" NR1 mice (fNR1). Amygdala microinjection of rAAV-GFP-Cre produced a decrease in NR1 gene expression and protein immunolabeling in postsynaptic sites of neurons without signs of compromised ultrastructural neuronal morphology. Amygdala NR1 gene deletion also did not affect locomotor, somatosensory, or sensory-motor behaviors. In addition, bilateral local NR1 gene deletion did not impact somatic or visceral withdrawal symptoms precipitated by naloxone in morphine-dependent mice. However, there was a significant deficit in the expression of an opioid withdrawal-induced conditioned place aversion in mice with amygdala NR1 deletion. These results indicate that functional amygdala NMDA receptors are involved in aversive psychological Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access

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Superoxide produced by the enzyme NADPH oxidase mediates crucial intracellular signaling cascades in the mNTS, a brain region populated by catecholaminergic neurons, as well as astroglia that play an important role in autonomic function. The mechanisms mediating NADPH oxidase (phox) activity in the neural regulation of cardiovascular processes are incompletely understood, however the subcellular localization of superoxide produced by the enzyme is likely to be an important regulatory factor. We used immunogold electron microscopy to determine the phenotypic and subcellular localization of the NADPH oxidase subunits p47 phox , gp91 phox, and p22 phox in the mNTS. The mNTS contains a large population of neurons that synthesize catecholamines. Significantly, catecholaminergic signaling can be modulated by redox reactions. Therefore, the relationship of NADPH oxidase subunit labeled neurons or glia with respect to catecholaminergic neurons was also determined by dual labeling for the superoxide producing enzyme and tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. In the mNTS, NADPH oxidase subunits were present primarily in somatodendritic processes and astrocytes, some of which also contained TH, or were contacted by TH labeled axons, respectively. Immunogold quantification of NADPH oxidase subunit localization showed that p47 phox and gp91 phox were present on the surface membrane, as well as vesicular organelles characteristic of calcium storing smooth endoplasmic reticula in dendritic and astroglial processes. These results indicate that NADPH oxidase assembly and consequent superoxide formation are likely to occur near the plasmalemma, as well as on vesicular organelles associated with intracellular calcium storage within mNTS neurons and glia. Thus, NADPH oxidase-derived superoxide may participate in intracellular signaling pathways linked to calcium regulation in diverse mNTS cell types. Moreover, NADPH oxidase-derived superoxide in neurons and glia may directly or indirectly modulate catecholaminergic neuron activity in the mNTS.

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