SUMMARY: In 2017, 19.7 million American adults struggled with substance use disorders, and in 2020, nearly 70,000 people died from opioid-involved overdoses. In opioid dependent individuals, environmental stress perpetuates cravings, and relapse is associated with strong stress responses and reactivity to drug cues. Unexpected reward and aversive stimuli affect ventral tegmental area (VTA) dopamine (DA) neuron activity, and studies neuropharmacological studies have revealed DA released from VTA in dorsal hippocampus (CA1) regulating contextual learning. Locus coeruleus noradrenergic (LC-NE) neurons may also release DA, which has the potential to affect contextual memory and plasticity in CA1. To address how aversive and rewarding contexts affect the opioid seeking, we must understand the neuropharmacological properties of norepinephrine (NE) and DA in opioid-associated contextual memory. The initial process of forming memories is acquisition, while after memories are reactivated, they are considered versatile before being reconsolidated for long term memory. While we know noradrenergic and dopaminergic receptors have been implicated in opioid contextual processing, yet there have not yet been clear pharmacological approaches examining endogenous monoamine release during contextual behavior. The central hypothesis of this proposal is that DA in CA1 is primarily released from the VTA, and only VTA-DA is necessary for, and LC-NE only enhances, memory acquisition and reconsolidation. Aim 1A uses two-photon ex vivo imaging and optogenetics with monoamine sensors to determine spatiotemporal properties of DA release from VTA terminals in dCA1, while Aim 1B will determine spatiotemporal properties of NE and DA release from LC in CA1. Using ex vivo pharmacology, we will elucidate which dopaminergic and noradrenergic mechanisms in CA1 influence the release of DA and NE from their source regions. Aim 2 will tests the necessity of NE and DA sources for morphine or naloxone place preference reconsolidation and the necessity of NE and DA for preference acquisition. In Aim 2A, we will simultaneously measure NE and DA dynamics as we pharmacologically inhibit their soma using chemogenetics to determine the necessity of VTA and LC in memory reconsolidation of a conditioned place preference or aversion. In Aim 2B, we will test the necessity of NE and DA for memory acquisition of conditioned preference or aversion by using CRISPR gene-editing to knockdown Dbh (NE-producing enzyme) in LC and Th (DA-producing enzyme) in VTA. This series of in vitro and in vivo neuropharmacology experiments will elucidate monoamine sufficiency and necessity in contextual processing to further our understanding of the influence of context in opioid seeking. This proposal will serve as training in neuropharmacology, two-photon optical imaging, optogenetics, chemogenetics, gene-editing, and behavioral pharmacology approaches. Ultimately, the training outlined in this proposal will enhance...