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

 描述（由申请人提供）：肥胖症通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质参与能量稳态的调节。多巴胺在这方面是一个很大的兴趣，因为它促进食物摄入，但它在能量稳态中的作用是复杂的，它通常被认为是中枢神经系统奖励系统的一部分。在这里，我们测试的一般假设，多巴胺能轴突支配下丘脑弓状核，并直接调节食物摄入量和体重，通过调节两个关键的神经元类型的活动在这里，但在相反的方向。弓状核在生理稳态，特别是能量稳态中起关键作用。弓状核神经元的两种关键类型是促食欲的阿黑皮素原（POMC）和促食欲的刺鼠相关肽（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，这些数据显示了多巴胺轴突对POMC和AgRP神经元的强神经支配，以及对POMC神经元的强多巴胺介导的抑制，但对相邻的AgRP神经元的兴奋。第一个目的是研究多巴胺轴突和POMC和AgRP神经元之间的相互作用的结构基板。我们测试的假设，多巴胺轴突与POMC或AgRP神经元使用共聚焦扫描激光显微镜和双免疫标记电子显微镜与抗多巴胺的抗血清，以确定多巴胺轴突的直接突触连接。为了检验向ARC的多巴胺投射来自腹侧被盖区（VTA）或其他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的控制下表达Cre重组酶的转基因小鼠的组合，结合脑内集中注射含有floxed的病毒载体，终止GFP或tdTomato报告基因以研究从多巴胺能细胞到弓状核的传出投射。我们还将采用注射Cre重组酶依赖性Brainbow型伪狂犬病病毒的弓状核;逆行轴突运输后，PRV通常在感染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。目的2验证多巴胺对POMC和AgRP神经元的作用是相反的，抑制POMC细胞，但兴奋AgRP神经元，这两种作用都促进了食物的摄入。我们将采用转基因小鼠表达各种报告基因，和全细胞电压和电流钳记录下丘脑脑切片。全细胞记录将使我们能够解决多巴胺作用于POMC和AgRP细胞的不同机制。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，以及表达氧化终止通道视紫红质-2（ChR 2）或ChIEF的病毒载体。蓝光激活ChR 2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。这种方法将被用于脑切片测试的假设，光激活释放的多巴胺轴突的递质将POMC和AgRP神经元上施加相反的效果。在小鼠体内实验中，我们测试的假设，从多巴胺轴突的光激活的递质释放将提高食物的摄入量。在多巴胺轴突的蓝光刺激期间和对照期间，将监测食物摄入、体重和活动。总之，这些实验将详细研究收敛的结构，跟踪，电生理和行为分析的假设，多巴胺轴突支配弓状POMC和AgRP神经元发挥积极的作用，在能量稳态。随着健康问题与不断增长的肥胖水平在这个国家，据估计达到30%的成年人口，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减少肥胖的趋势。

 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.