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

 描述(申请人提供)：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、癌症和过早死亡，已成为美国的主要健康问题之一。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。多巴胺是这方面的一个很大的兴趣，因为它促进食物的摄取，但它在能量平衡中的作用是复杂的，它经常被认为是中枢神经系统奖励系统的一部分。在这里，我们测试了一个普遍的假设，即多巴胺能轴突支配下丘脑弓状核，并通过调节两种关键神经元类型的活动直接调节食物摄入量和体重，但方向相反。弓状核在生理内稳态，特别是能量内稳态中起着关键作用。弓状核神经元的两种关键类型是厌食性阿片黑素原(POMC)神经元和食欲性刺鼠相关肽(AgRP)/神经肽Y(NPY)神经元。我们的初步数据表明，多巴胺轴突对POMC和AgRP神经元有很强的神经支配作用，对POMC神经元有强烈的多巴胺中介抑制作用，但对邻近的AgRP神经元有兴奋作用。第一个目的是研究多巴胺轴突与POMC和AgRP神经元相互作用的结构底物。我们用共聚焦激光扫描显微镜和双重免疫标记电子显微镜结合多巴胺抗血清来验证多巴胺轴突与POMC或AgRP神经元直接突触联系的假说。为了验证多巴胺投射到ARC的假设来自腹侧被盖区(VTA)或其他多巴胺能群体，我们将使用在酪氨酸羟基酶或多巴胺转运体启动子控制下表达Cre重组酶的转基因小鼠的组合，并在脑内聚焦注射含有Flobled-Stop GFP或tdTomato报告基因的病毒载体，以研究多巴胺能细胞向弓状核的传出投射。我们还将使用向弓状核注射依赖Cre重组酶的脑弓型伪狂犬病病毒；在逆行轴突运输后，PRV在感染细胞中通常表达红色荧光报告，但在表达Cre的多巴胺细胞中，颜色改变为黄色或蓝色。目的2验证多巴胺对POMC和AgRP神经元起相反作用的假说，抑制POMC细胞，但兴奋AgRP神经元，两者都增加摄食量。我们将使用表达各种报告基因的转基因小鼠，并在下丘脑脑片上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，与表达FLOXED-STOP通道视紫红质-2(ChR2)或Head的病毒载体相结合。蓝光激活ChR2或主要介导的内向电流，允许我们选择性地光刺激弓状核内的多巴胺轴突。这种方法将被用在脑片上来检验这样的假设，即光激活的多巴胺轴突递质释放将对POMC和AgRP神经元产生相反的影响。在小鼠的活体实验中，我们测试了从多巴胺轴突释放的光激活递质将增加食物摄入量的假设。在蓝光刺激多巴胺轴突期间和对照期间，将监测食物摄入量、体重和活动。总之，这些实验将结合结构、追踪、电生理和行为分析详细检验这一假说，即支配弓状POMC和AgRP神经元的多巴胺轴突在能量平衡中发挥积极作用。据估计，美国30%的成年人口与肥胖相关的健康问题，以及相关的健康并发症，了解和了解其中涉及的脑细胞将有助于找到减少肥胖趋势的新方法。

 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.