Project Summary: Nociceptin/orphanin FQ (N/OFQ) has continued to gain increasing recognition as an important player in the central control of biological process such as addiction, energy homeostasis and reproduction. It exerts these actions via stimulation of its cognate nociceptin opioid (NOP) receptor. The overall goal of this proposal is to gain a better understanding of the NOP receptor-mediated regulation of energy balance via the homeostatic and hedonic circuitries, and the cellular mechanism(s) by which N/OFQ affects the excitability of anorexigenic proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC), of anorexigenic pituitary adenylyl cyclase-activating polypeptide (PACAP) neurons in the hypothalamic ventromedial nucleus (VMN), and of mesolimbicortical (A10) dopamine neurons in the ventral tegmental area (VTA) that encode natural- and drug-induced reward. The fundamental hypothesis is that N/OFQ regulates appetitive behavior and metabolism in a diet- and sex- dependent fashion that can be differentially influenced by the stage of the estrous cycle. It does so, in large part, via pleiotropic inhibitory actions involving direct NOP receptor-mediated activation of G protein-gated, inwardly-rectifying (GIRK) channels in POMC, PACAP and A10 dopamine neurons, and presynaptic inhibition of excitatory neurotransmission at synapses formed between upstream PACAP neurons and downstream POMC neurons. NOP receptor-mediated regulation of energy homeostasis and excitability of POMC, PACAP and A10 dopamine neurons will be studied using transgenic N/OFQ-cre, N/OFQ-cre/eGFP-POMC, PACAP-cre/eGFP POMC PACAP-cre, and tyrosine hydroxylase (TH)-cre mice models, in which either channelrhodpsin-2 or a Gq-coupled M3 or designer receptor activated exclusively by designer drugs is delivered via a viral vector construct into the ARC, VMN or VTA and integrated into neurons expressing PACAP, TH or N/OFQ for in vivo and in vitro optogenetic and chemogenetic experimentation. For certain in vitro electrophysiological experiments, exogenous N/OFQ will be strategically bath applied. In Specific Aim 1, I will evaluate whether long-term exposure to a "Westernized" high-fat diet differentially alters N/OFQ sensitivity in males and cycling females by assessing how it influences the capability of exogenous N/OFQ administration, as well as endogenous N/OFQ release, in the ARC and VMN within the homeostatic energy balance circuitry to increase energy intake and decrease energy expenditure via pleiotropic actions at VMN PACAP/ARC POMC synapses. In Specific Aim 2, the objective is to determine whether differential high-fat diet-induced alterations in N/OFQ sensitivity give rise to equally disparate changes within the hedonic energy balance circuitry. Specifically, I will evaluate whether high-fat diet exposure differentially enhances the capability of exogenous and endogenous N/OFQ to directly inhibit A10 dopamine neurons via NOP receptor-mediated activation...