Project Summary: The primary goal of this training proposal is to dissect the neurocircuitry of medial nucleus accumbens shell (mNAcSh) dynorphin and enkephalin neurons in reward processing. By local interconnectivity and through output projections to lateral hypothalamus (LH), ventral pallidum (VP) and ventral tegmental area (VTA), these neuronal populations may process reward value and orchestrate activity of downstream limbic circuitry to coordinate consummatory behavior and reinforcement. The first aim of this training proposal seeks to characterize the neural activity of dynorphin and enkephalin neuronal populations in mNAcSh during the anticipation and consumption of reward. Aim 1A uses in vivo 2-photon microscopy to observe the endogenous activity of dynorphin and enkephalin neurons with single cell resolution during consumption of sucrose reward of varying concentrations, while Aim 1B leverages the same approach to observe endogenous neural activity during cue- reward associative learning. Aim 2A seeks to determine the molecular identity of these mNAcSh neurons that project downstream to LH, VP and VTA through retrograde tracing techniques and fluorescent in situ hybridization. Aim 2B uses multisite in vivo fiber photometry to simultaneously record the neural activity of mNAcSh neuron axonal terminals in LH, VP and VTA during the consumption of sucrose reward of varying concentrations and during cue-reward associative learning. Aim 3 leverages time-locked optogenetic manipulations to establish necessity and sufficiency of observed endogenous neural activity of mNAcSh axonal terminals in LH, VP and VTA in determining rate of sucrose consumption and rate of cue-reward associative learning. This will be the first study to use in vivo calcium imaging in mNAcSh, fiber photometry of mNAcSh output projections and time-locked optogenetic manipulation of these circuits to further understand the role of this brain region and its output circuitry in reward processing. Leveraging new, high-resolution approaches will allow us to further understand this critical endogenous opioid system and to identify potential mechanisms that underlie addiction. During my training period, I will learn novel optical imaging, optogenetic, tracing and behavioral techniques, as well as pursue my specific career development plan to prepare me for independence as an academic researcher.