Dopamine Excites Orexigenic AgRP/NPY Neurons, but Inhibits Anorexic POMC Neurons

多巴胺兴奋促食欲 AgRP/NPY 神经元，但抑制厌食 POMC 神经元

• 批准号: 8888338
• 负责人: ANTHONY N VAN DEN POL
• 金额: $ 37.46万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8888338
• 关键词: Address; Adult; Attention; Axon; Axonal Transport; Behavior; Behavioral; Body Weight; Brain; Cells; Cessation of life; Code; Color; Complement; Complex; Corpus striatum structure; Country; Coupled; Data; Diabetes Mellitus; Dopamine; Dopaminergic Cell; Dorsal; Eating; Electron Microscopy; Electrophysiology (science); Feedback; Fluorescence; Genes; Health; Heart Diseases; Homeostasis; Hypothalamic structure; Immune Sera; In Vitro; Injection of therapeutic agent; Label; Laser Scanning Confocal Microscopy; Lateral; Life; Light; Malignant Neoplasms; Measures; Mediating; Midbrain structure; Monitor; Mus; Neuromodulator; Neurons; Neuropeptides; Neurotransmitters; Nucleus Accumbens; Obesity; Pattern; Peptides; Phenotype; Play; Population; Pro-Opiomelanocortin; Regulation; Reporter; Reporter Genes; Rewards; Role; Secondary to; Slice; Stroke; Structure of nucleus infundibularis hypothalami; Substrate Interaction; Suid Herpesvirus 1; Synapses; System; Testing; Transgenic Mice; Tyrosine 3-Monooxygenase; Ventral Tegmental Area; Viral Vector; Whole-Cell Recordings; brain cell; dopamine transporter; dopaminergic neuron; energy balance; hedonic; immunocytochemistry; in vivo; interest; nerve supply; neuropeptide Y; novel; novel strategies; optogenetics; patch clamp; promoter; public health relevance; receptor; recombinase; research study; response; trend; voltage; voltage clamp

 DESCRIPTION (provided by applicant): Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become one of the primary health concerns in the US. Many neurotransmitters and neuromodulators in the CNS participate in the regulation of energy homeostasis. Dopamine is a great interest in this regard because it promotes food intake, but its role in energy homeostasis is complex and it is often considered as part of a CNS reward system. Here we test the general hypothesis that dopaminergic axons innervate the hypothalamic arcuate nucleus and directly modulate food intake and body weight by regulating the activity of two key neuron types here, but in opposite directions. The arcuate nucleus plays a key role in physiological homeostasis, and particularly energy homeostasis. Two key types of arcuate nucleus neurons are the anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP)/neuropeptide Y (NPY) neurons. The studies proposed below are supported by our preliminary data showing strong innervation of POMC and AgRP neurons by dopamine axons, and by a robust dopaminemediated inhibition of POMC neurons, but excitation of neighboring AgRP neurons. The first Aim examines the structural substrates for interaction between dopamine axons and the POMC and AgRP neurons. We test the hypothesis that dopamine axons make direct synaptic connections with POMC or AgRP neurons using confocal scanning laser microscopy and dual immunolabel electron microscopy with antisera against dopamine to identify dopamine axons. To test the hypothesis that dopamine projections to the ARC arise from the ventral tegmental area (VTA) or other dopaminergic populations, we will use a combination of transgenic mice expressing Cre recombinase under control of the tyrosine hydroxylase or dopamine transporter promoter, coupled with focused intracerebral injections of viral vector containing floxed-stop GFP or tdTomato reporter genes to study efferent projections to the arcuate nucleus from dopaminergic cells. We will also employ injection into the arcuate nucleus of a Cre recombinase-dependent Brainbow-type pseudorabies virus; after retrograde axonal transport the PRV normally expresses a red fluorescent reporter in infected cells, but in dopamine cells expressing Cre, changes color to yellow or blue. Aim 2 tests the hypothesis that dopamine exerts opposing actions on POMC and AgRP neurons, inhibiting POMC cells, but exciting AgRP neurons, both actions enhancing food intake. We will employ transgenic mice expressing various reporter genes, and whole cell voltage- and current clamp recording in hypothalamic brain slices. Whole cell recording will allow us to address different mechanisms of dopamine actions on the POMC and AgRP cells. In Aim 3, a final set of experiments employs optogenetics and transgenic mice expressing Cre recombinase in dopamine neurons, coupled with viral vectors expressing floxed-stop channelrhodopsin-2 (ChR2) or ChIEF. Blue light activates a ChR2- or ChIEF-mediated inward current, allowing us to photostimulate selectively the dopamine axons within the arcuate nucleus. This approach will be used in brain slices to test the hypothesis that light-activated release of transmitter from dopamine axons will exert opposite effects on POMC and AgRP neurons. In mouse in vivo experiments, we test the hypothesis that light-activated transmitter release from dopamine axons will enhance food intake. Food intake, body weight, and activity will be monitored during periods of blue light stimulation of the dopamine axons, and during control periods. Together, these experiments will examine in detail with converging structural, tracing, electrophysiological, and behavioral analyses the hypothesis that dopamine axons innervating arcuate POMC and AgRP neurons play a positive role in energy homeostasis. With the health problems associated with the growing levels of obesity in this country, by some estimates reaching 30% of the adult population, and the associated health complications, knowing and understanding the brain cells involved will help to identify novel approaches to reducing the trend toward obesity.

 描述（由申请人提供）：肥胖常常导致继发性健康并发症，包括心脏病、糖尿病、中风、癌症和早逝，已成为美国主要的健康问题之一。中枢神经系统中的许多神经递质和神经调节剂参与能量稳态的调节。多巴胺在这方面引起了很大的兴趣，因为它可以促进食物摄入，但它在能量稳态中的作用很复杂，并且通常被认为是中枢神经系统奖励系统的一部分。在这里，我们测试了一般假设，即多巴胺能轴突支配下丘脑弓状核，并通过调节此处两种关键神经元类型的活动来直接调节食物摄入和体重，但方向相反。弓状核在生理稳态，特别是能量稳态中起着关键作用。弓状核神经元的两种关键类型是促食欲阿黑皮素原 (POMC) 和促食欲刺鼠相关肽 (AgRP)/神经肽 Y (NPY) 神经元。下面提出的研究得到了我们的初步数据的支持，这些数据显示多巴胺轴突对 POMC 和 AgRP 神经元的强烈神经支配，以及对 POMC 神经元的强大多巴胺介导的抑制，但对邻近 AgRP 神经元的兴奋。第一个目标检查多巴胺轴突与 POMC 和 AgRP 神经元之间相互作用的结构底物。我们使用共焦扫描激光显微镜和双免疫标记电子显微镜以及抗多巴胺抗血清来验证多巴胺轴突与 POMC 或 AgRP 神经元建立直接突触连接的假设，以识别多巴胺轴突。为了检验多巴胺投射到 ARC 源自腹侧被盖区 (VTA) 或多巴胺能群体的假设，我们将使用在酪氨酸羟化酶或多巴胺转运蛋白启动子控制下表达 Cre 重组酶的转基因小鼠组合，并结合脑内集中注射含有 floxed-stop GFP 或 tdTomato 报告基因用于研究多巴胺能细胞向弓状核的传出投射。我们还将采用 Cre 重组酶依赖性 Brainbow 型伪狂犬病病毒注射到弓形核中；逆行轴突运输后，PRV 通常在受感染的细胞中表达红色荧光报告基因，但在表达 Cre 的多巴胺细胞中，颜色变为黄色或蓝色。目标 2 检验了这样的假设：多巴胺对 POMC 和 AgRP 神经元发挥相​​反的作用，抑制 POMC 细胞，但刺激 AgRP 神经元，这两种作用都会增加食物摄入量。我们将使用表达各种报告基因的转基因小鼠，并在下丘脑脑切片中进行全细胞电压和电流钳记录。全细胞记录将使我们能够解决多巴胺对 POMC 和 AgRP 细胞作用的不同机制。在目标 3 中，最后一组实验采用光遗传学和在多巴胺神经元中表达 Cre 重组酶的转基因小鼠，以及表达 floxed-stop 通道视紫红质-2 (ChR2) 或 ChIEF 的病毒载体。蓝光激活 ChR2 或 ChIEF 介导的内向电流，使我们能够选择性地光刺激弓状核内的多巴胺轴突。这种方法将用于脑切片，以测试多巴胺轴突的光激活递质释放将对 POMC 和 AgRP 神经元产生相反影响的假设。在小鼠体内实验中，我们测试了多巴胺轴突释放的光激活递质将增加食物摄入的假设。在蓝光刺激多巴胺轴突期间和控制期间将监测食物摄入量、体重和活动。这些实验将结合结构、示踪、电生理学和行为分析，详细检验以下假设：支配弓状 POMC 和 AgRP 神经元的多巴胺轴突在能量稳态中发挥积极作用。在这个国家，肥胖率不断上升，导致了健康问题，据估计，肥胖率达到了成年人口的 30%，以及相关的健康并发症，了解和理解所涉及的脑细胞将有助于找到减少肥胖趋势的新方法。