Role of the Lateral Hypothalamic Area in Leptin Action

描述（由申请人提供）：这是NIH 5R01DK078056在NOT-OD-09-058项下的竞争性修订申请，“NIH宣布竞争性修订申请的恢复法案资金可用性”。肥胖在美国的持续流行代表了一种公共卫生紧急情况，仍然没有特定的治疗方法。为了设计预防和治疗肥胖的具体治疗方法，我们必须首先了解调节进食和能量消耗的机制。在本次竞争性修订申请的母奖“外侧下丘脑区在瘦素作用中的作用”中，我们定义并正在研究外侧下丘脑区（LHA）中表达leprb的新型含有神经紧张素（Nts）的神经元。虽然Nts和LepRb在整个中枢神经系统的许多其他位置都有表达，但它们的共表达（或交叉，如维恩图所示）仅限于这些Nts/LHA LepRb神经元。因此，本修订申请的父母奖提出，通过使用标准的Cre/LoxP技术（即Ntscre和Leprfl）从这些神经元中删除LepRb，研究瘦素通过Nts/LHA LepRb神经元的作用。到目前为止，我们的研究结果表明，Nts/LHA LepRb神经元调节中边缘DA系统中的多巴胺（DA）含量，并控制摄食和活动。除了表达Nts外，Nts/LHA LepRb神经元还含有抑制性神经递质GABA。GABA神经元在整个大脑中的广泛分布（包括许多LepRb群体）阻止了通过标准Cre/LoxP技术分析Nts/LHA LepRb神经元的GABA作用。Nts/LHA LepRb神经功能的其他方面同样不可能单独使用Cre/LoxP进行研究。竞争修订的目的：利用Flp/Frt和Cre/LoxP相结合的方法激活和灭活同时表达LepRb和Nts的神经元中感兴趣的基因。(1)制备在LepRb神经元中表达Flp重组酶的LeprFlp小鼠。我们将使用LeprFlp与我们现有的Ntscre结合，通过Cre？我们正在生产的可诱导的玫瑰位点转基因。(2)我们将生成NtsNeo-Cre小鼠，其中Flp（来自LeprFlp）去除转录阻断的first - neo盒体，诱导表达nts的LepRb神经元中Cre的表达。因此，该系统将允许在Nts/LHA LepRb神经元中特异性地删除标准条件等位基因（例如Vgatfl）。我们将利用这些“交叉”遗传技术更深入地探索Nts/LHA LepRb神经元在能量稳态中的作用。我们由此获得的信息将为瘦素作用和能量平衡的机制提供重要的见解。当我们试图确定持续流行的肥胖的发病机制和潜在的治疗靶点时，这种理解是至关重要的。此外，这些研究将验证一套新的工具，可以从基因上探测大脑（或其他）细胞的交叉子集。

Description (provided by applicant): 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.