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: 9213370
• 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/9213370
• 关键词: Address; Adult; Appetite Stimulants; Attention; Axon; Axonal Transport; Behavior; Behavioral; Body Weight; Brain; Cells; Cessation of life; Closure by clamp; Code; Color; Complement; Complex; Corpus striatum structure; Country; Coupled; Data; Diabetes Mellitus; Dopamine; Dopaminergic Cell; Dorsal; Eating; Electron Microscopy; Electrophysiology (science); Enterobacteria phage P1 Cre recombinase; Feedback; Fluorescence; Genes; Health; Heart Diseases; Homeostasis; Hypothalamic structure; Immune Sera; In Vitro; Injection of therapeutic agent; Label; Laser Scanning Confocal Microscopy; Lateral; Light; LoxP-flanked allele; Malignant Neoplasms; Measures; Mediating; Midbrain structure; Monitor; Morphology; 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; Synapses; System; Testing; Transgenic Mice; Tyrosine 3-Monooxygenase; Ventral Tegmental Area; Viral Vector; Whole-Cell Recordings; brain cell; dopamine transporter; dopaminergic neuron; energy balance; experimental study; hedonic; immunocytochemistry; in vivo; interest; mad itch virus; nerve supply; neuropeptide Y; novel; novel strategies; optogenetics; patch clamp; promoter; public health relevance; receptor; 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（适用提供）：肥胖通常会导致继发性健康并发症，包括心脏病，糖尿病，中风，癌症和早期死亡，已成为美国的主要健康问题之一。中枢神经系统参与能量稳态调节的许多神经递质和神经调节剂。多巴胺在这方面非常感兴趣，因为它可以促进食物摄入量，但其在能量稳态中的作用很复杂，通常被视为CNS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弧形核，并通过在此处重新调节两种关键神经元类型的活性，但在相反的方向上直接调节食物摄入和体重。弧形核在物理体内稳态，尤其是能量体内稳态中起关键作用。两种关键类型的弓形核神经元是厌食性肌蛋白酶素（POMC）和甲状腺素Agouti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支持，该数据表明多巴胺轴突对POMC和AGRP神经元的强大神经支配，以及强大的多巴胺介导的POMC神经元的抑制作用，但对相邻的AGRP神经元的兴奋。第一个目标检查了多巴胺轴突与POMC和AGRP神经元之间相互作用的结构底物。我们测试了多巴胺轴突使用共聚焦扫描激光显微镜和双重免疫标记电子显微镜与抗抗激素相对于多巴胺鉴定多巴胺轴突的，多巴胺轴突与POMC或AGRP神经元直接与POMC或AGRP神经元进行了直接突触连接。 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报告基因研究对多巴胺能细胞的弓形核的有效投影。我们还将使用注射到CRE重组酶依赖性脑弓型伪型病毒的弧形核中；逆行轴突转运后，PRV通常在感染细胞中表达红色荧光报告基因，但是在表达CRE的多巴胺细胞中，颜色变为黄色或蓝色。 AIM 2检验了多巴胺对POMC和AGRP神经元的相反作用，抑制POMC细胞但令人兴奋的AGRP神经元的假设，这两种动作都可以增强食物摄入量。我们将采用表达各种报告基因的转基因小鼠，以及下丘脑脑切片中的全细胞电压和电流夹具记录。全细胞记录将使我们能够解决多巴胺在POMC和AGRP细胞上的不同机制。在AIM 3中，一组最终的实验员工在多巴胺神经元中表达CRE重组酶的转基因小鼠，并与表达Floxed-Stop-Stop ChannelRhopopsin-2（Chr2）或首席的病毒载体结合。蓝光激活ChR2或主要介导的内向电流，使我们能够在弧形核中选择性地刺激多巴胺轴突。这种方法将用于大脑切片中，以测试以下假设：从多巴胺轴突中发射光的光激活将对POMC和AGRP神经元产生相反的影响。在小鼠体内实验中，我们检验了以下假设：从多巴胺轴突中释放出光激活的发射机将增强食物摄入量。在多巴胺轴突的蓝光刺激期间以及在控制期间，将监测食物摄入，体重和活动。总之，这些实验将通过收敛的结构，跟踪，电生理和行为分析详细研究，即在能量稳态中支配ARCAURE POMC和AGRP神经元的多巴胺轴突在能源稳态中起积极作用。随着与该国肥胖水平不断增长相关的健康问题，估计有些人占成年人口的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确定新的方法来降低肥胖趋势。