This is an application for competitive revision of NIH 5R01DK078056 under NOT-OD-09-058, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications."The ongoing epidemic of obesity in the United States represents a public health emergency that remains without specific therapy. To design specific treatments to prevent and treat obesity, we must first understand the mechanisms that regulate feeding and energy expenditure. In the parent award of this application for competitive revision, "Role of the lateral hypothalamic area in leptin action," we have defined and are studying novel LepRb-expressing neurotensin (Nts)-containing neurons in the lateral hypothalamic area (LHA). While Nts and LepRb are each expressed in numerous other locations throughout the CNS, their co-expression (or intersection, as in a Venn diagram) is restricted to these Nts/LHA LepRb neurons. The parent award for this revision application thus proposes to study the action of leptin via Nts/LHA LepRb neurons by deleting LepRb from these neurons using standard Cre/LoxP technology (i.e., Ntscre and Leprfl). Our findings to date suggest that Nts/LHA LepRb neurons modulate dopamine (DA) content in the mesolimbic DA system, and also control feeding and activity. In addition to expressing Nts, Nts/LHA LepRb neurons contain the inhibitory neurotransmitter, GABA. The wide distribution of GABA neurons throughout the brain (including in numerous LepRb populations) prevents the analysis of GABA action via Nts/LHA LepRb neurons by standard Cre/LoxP technology. Other aspects of Nts/LHA LepRb neural function are similarly not possible to study using Cre/LoxP alone. This application for competitive revision of the parent grant thus seeks to: Aim of Competitive Revision: Utilize combined Flp/Frt and Cre/LoxP approaches to activate and deactivate genes of interest in neurons that express both LepRb and Nts. (1) We will generate LeprFlp mice that express Flp recombinase in LepRb neurons. We will use LeprFlp in combination with our existing Ntscre to activate gene expression via a Cre?inducible Rosa locus transgene that we are producing. (2) We will generate NtsNeo-Cre mice, in which Flp (as from LeprFlp) removes a transcription-blocking Frt-Neo cassette to induce the expression of Cre in Nts-expressing LepRb neurons. This system will thus permit the deletion of standard conditional alleles (e.g. Vgatfl) specifically in Nts/LHA LepRb neurons. We will utilize these "intersectional" genetic technologies to more deeply explore the role of Nts/LHA LepRb neurons in energy homeostasis. The information that we will thereby obtain will provide important insight into mechanisms of leptin action and energy balance. This understanding is crucial as we seek to determine the pathogenesis of, and potential therapeutic targets for, the ongoing epidemic of obesity. Additionally, these studies will validate a novel set of tools that can genetically probe intersectional subsets of brain (or other) cells. PUBLIC HEALTH RELEVANCE: The ongoing epidemic of obesity in the United States represents a public health emergency that remains unchecked and without specific therapy. To design specific treatments to prevent and treat obesity, we must first understand the mechanisms that regulate feeding and energy expenditure in order to identify potential therapeutic targets. In this proposal, we will develop new transgenic animals to enable the more precise manipulation and study of specific subsets of neurons, including an important group of lateral hypothalamic neurons that contribute to body energy homeostasis. These studies will help us to define the function of these neurons and the potential therapeutic targets that they represent.