Arcuate nucleus glutamatergic neurons modulate energy homeostasis

弓状核谷氨酸能神经元调节能量稳态

• 批准号: 7889376
• 负责人: ANTHONY N VAN DEN POL
• 金额: $ 41.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-10 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7889376
• 关键词: Address; Adult; Affect; Animals; Attention; Axon; Body Weight; Brain; Brain region; Cell Nucleus; Cell physiology; Cells; Cessation of life; Characteristics; Cues; Diabetes Mellitus; Dyes; Eating; Electron Microscopy; Electrophysiology (science); Endocrine; Endocrine Glands; Epidemic; Excitatory Amino Acid Antagonists; Exhibits; Expenditure; Experimental Designs; Functional disorder; Gene Expression; Glucose; Glutamates; Growth; Health; Homeostasis; Hypothalamic structure; Joints; Label; Lactation; Leptin; Malignant Neoplasms; Membrane; Metabolism; Methods; Microinjections; Mus; Neuromodulator; Neurons; Neuropeptides; Neurosecretory Systems; Neurotransmitters; Obesity; Organ; Output; Paper; Peptides; Peripheral; Pituitary Gland; Play; Population; Pro-Opiomelanocortin; Property; Recombinants; Regulation; Reporter; Reporter Genes; Reproduction; Role; Series; Signal Transduction; Slice; Stimulus; Stress; Stroke; Structure of nucleus infundibularis hypothalami; Suid Herpesvirus 1; Synapses; Testing; Time; Transgenic Mice; Work; base; cost; energy balance; excitatory neuron; gamma-Aminobutyric Acid; hypertensive heart disease; immunocytochemistry; inhibitory neuron; insight; mind control; neuronal cell body; neuropeptide Y; obesity treatment; patch clamp; public health relevance; research study; response; vesicular glutamate transporter 2; voltage clamp

DESCRIPTION (provided by applicant): The underlying health problem that this application addresses is the growing epidemic of obesity that now affects 30% of the adult population, and the resultant increase in heart disease, hypertension, diabetes, joint dysfunction, stroke, cancer, and early death that is estimated to cost upwards of 75 billion dollars per year. Many factors contribute to the obesity problem today. The hypothalamic arcuate nucleus in the brain acts like the information hub of energy balance, receiving information both from peripheral organs involved in energy storage or release, and receiving axonal information from other regions of the brain that also play important roles in CNS regulation of energy homeostasis, and sending out efferent information that regulates food intake and utilization. A focus for many years in this field has been the neuropeptides involved in energy regulation, and the hypothalamic neurons that secrete them. Most of the critical peptides involved in the regulation of energy homeostasis have been colocalized with the inhibitory transmitter GABA in the arcuate nucleus. This application focuses on what appears to be a new cellular player in the CNS regulation of energy balance that we have identified, the arcuate glutamatergic neuron, a cell that has the profile of one that reduces food intake. In most other regions of the brain glutamate is recognized as a major neurotransmitter. But in the hypothalamus, relatively little attention has been given to glutamate neurons, despite the fact that in the presence of glutamate receptor antagonists there is virtually no excitatory synaptic activity in the arcuate nucleus, or elsewhere in the hypothalamus. Prior to submitting this application, we have solved a central problem, that of recognizing these glutamate cells that exhibit no morphological difference from other hypothalamic cells, by generating a transgenic mouse that expresses the reporter GFP under the control of the vesicular glutamate transporter 2 (vGluT2) selectively in glutamate neurons. Our experiments utilize a combination of whole cell patch clamp electrophysiology, tract tracing with fluorogold and pseudorabies virus, ultrastructural immunocytochemistry, and altered gene expression in the context of challenges to whole animal energy balance. The first set of experiments address the hypothesis that the glutamate neurons show the same efferent axonal projections as the inhibitory neurons of the arcuate nucleus. This will be tested with fluorogold and recombinant pseudorabies virus microinjections into putative target regions. To test the hypothesis that arcuate glutamate cells regulate the activity of anorexigenic proopiomelanocortin (POMC) neurons, we will record from POMC neurons while stimulating local glutamate cells with the excitatory microdrop method to activate cell bodies but not axons of passage. Parallel experiments address the question of whether glutamate cells innervate each other, thereby increasing the timing and power of their output. Ultrastructural dual label immunocytochemistry will be used to test the hypothesis that local orexigenic neuropeptide Y (NPY) immunoreactive axons make synaptic contact with the glutamate cells, similar to the NPY axons that synapse with the anorexigenic POMC neurons. A second set of experiments, using whole cell patch clamp recording in hypothalamic slices, addresses the question of "What active or passive membrane characteristics make the glutamate neurons unique", compared with the GABAergic neurons of the arcuate nucleus that have received substantial attention. A third set of electrophysiological experiments tests the hypothesis that neuropeptides released from other arcuate nucleus neurons involved in the regulation of energy homeostasis modulate the activity of the arcuate glutamate neurons. In the fourth set of experiments, we ask whether arcuate glutamate neurons respond to long distance cues relating to energy homeostasis, particularly glucose and leptin. Together, these experiments will reveal the organization and cellular actions and responses of a unique and previously uncharacterized excitatory neuron in the arcuate nucleus. Understanding these glutamatergic cells should give us a better appreciation of the cellular mechanisms underlying energy homeostasis and body weight regulation, and should give us new insight into the potential treatment of obesity through those neurons that control food intake and expenditure. Many neurons in the arcuate nucleus have multiple roles; it is possible that the glutamate cell is no exception, and may play a role in other functions that this small but critical part of the brain controls, including regulation of the pituitary and other endocrine organs, reproduction and lactation, growth, metabolism, and response to stress. PUBLIC HEALTH RELEVANCE: The growing epidemic of obesity, found in about 30% of the adults in the USA, has led to an increase in heart disease, hypertension, diabetes, stroke, cancer, and early death that is estimated to cost upwards of 75 billion dollars per year. Previous work on how the brain controls energy homeostasis and body weight has focused mostly on inhibitory peptidergic neurons that use the neurotransmitter GABA. In addition the hypothalamic arcuate nucleus plays a key role in pituitary control, endocrine regulation, reproduction, and metabolism. We will focus on a newly discovered neuron in the hypothalamic arcuate nucleus that is excitatory and uses glutamate as a neurotransmitter. Based on how the neuron responds to a number of stimuli studied in preliminary work, we will test the hypothesis that these cells may function to reduce body weight by local excitation of other neurons that are known to reduce food intake and body weight, and by inhibition from cells that increase food intake. Understanding this cell and how it responds to signals of food intake, and what other cells it regulates should allow a new avenue to intervene in how the brain controls body weight, and ultimately may serve to enhance our ability to reverse the obesity epidemic. Similar to other neurons of the arcuate nucleus that serve multiple homeostatic functions, the function of the excitatory arcuate glutamate neuron may include other roles in addition to that of energy homeostasis.

描述（由申请人提供）：本申请解决的潜在健康问题是肥胖的流行病现在影响了30％的成年人口，以及导致的心脏病，高血压，糖尿病，糖尿病，关节功能障碍，中风，癌症和早期死亡的增加，估计每年成本超过750亿美元。 许多因素导致了当今的肥胖问题。大脑中的下丘脑弧形核的作用就像能量平衡的信息中心，从参与能量储存或释放的外围器官中接收信息，并从大脑其他区域接收轴突信息，在CNS调节能量稳态中也起着重要作用，并传递出富有的信息，以调节食物的富裕信息。在该领域多年的重点是参与能量调节的神经肽以及分泌它们的下丘脑神经元。参与能量稳态调节的大多数关键肽已与弓形核中的抑制性发射器GABA共定位。该应用的重点是我们确定的CNS能量平衡调节中似乎是新的细胞播放器，即弧形谷氨酸能神经元，谷氨酸能神经元具有减少食物摄入的细胞。在大多数其他区域，脑谷氨酸盐被认为是主要的神经递质。但是在下丘脑中，尽管在存在谷氨酸受体拮抗剂的情况下，对谷氨酸神经元的关注很少，但在弧形核中几乎没有兴奋性突触活性，或者在下丘脑的其他地方没有兴奋性突触活性。在提交此应用之前，我们解决了一个核心问题，即识别这些谷氨酸细胞与其他下丘脑细胞没有形态学差异，通过产生一种转基因小鼠在囊泡谷氨酸转运蛋白2（vglut2）中表达报告基因GFP的转基因小鼠在Glutamate神经元中选择性地选择性地表达了Reporter GFP。 我们的实验利用了全细胞斑块夹电生理学，与荧光质量和伪标记病毒，超微结构免疫细胞化学和基因表达改变基因表达的结合，在对整个动物能量平衡的挑战的背景下改变了基因表达。 第一组实验解决了以下假设：谷氨酸神经元表现出与弧形核的抑制性神经元相同的传出轴突投射。这将通过荧光岩和重组假性病毒对假定的靶区域进行测试。为了测试抗谷氨酸细胞的假说调节厌食性肌蛋白酶皮质素（POMC）神经元的活性，我们将从POMC神经元中记录，同时用兴奋性的微角色方法刺激局部谷氨酸细胞，以激活细胞体，而不是轴突。并行实验解决了谷氨酸细胞是否相互支配的问题，从而增加了其输出的时间和功率。超微结构双重标记免疫细胞化学将用于检验以下假设：局部甲状腺素神经肽Y（NPY）免疫反应性轴突与谷氨酸细胞具有突触接触，类似于与厌食性POMC神经元突触的NPY轴突。 与下丘脑切片中的全细胞贴片夹记录的第二组实验有关“哪种活性或被动膜特征使谷氨酸神经元具有独特之处”，与弧形核的Gabaergic神经元相比，谷氨酸神经元独特”。 第三组电生理实验检验了从其他弧形神经元中释放出参与能量稳态调节的神经肽可调节弧形谷氨酸神经元的活性。 在第四组实验中，我们询问弧形神经元是否应对与能量稳态有关的长距离提示，尤其是葡萄糖和瘦素。 这些实验将共同揭示弓形核中独特且先前未表征的兴奋性神经元的组织和细胞作用和反应。了解这些谷氨酸能细胞应该使我们更好地理解能量稳态和体重调节的细胞机制，并应通过控制食物摄入和支出的那些神经元对肥胖的潜在治疗进行新的了解。弓形核中的许多神经元具有多个作用。谷氨酸细胞可能也不例外，并且可能在其他功能中发挥作用，大脑控制的这个小但关键的部分，包括调节垂体和其他内分泌器官，繁殖和泌乳，生长，代谢以及对压力的反应。 公共卫生相关性：在美国约30％的成年人中发现的肥胖流行流行已导致心脏病，高血压，糖尿病，中风，癌症和早期死亡的增加，估计每年成本高达750亿美元。先前关于大脑如何控制能量稳态和体重的工作主要集中在使用神经递质GABA的抑制性肽基神经元上。另外，下丘脑弧形核在垂体控制，内分泌调节，繁殖和代谢中起关键作用。我们将专注于下丘脑弓形核中新发现的神经元，该神经元具有兴奋性，并将谷氨酸用作神经递质。基于神经元对初步工作中研究的许多刺激的反应，我们将测试以下假设：这些细胞可能通过局部激发其他神经元的兴奋来减轻体重，这些神经元通过降低食物摄入和体重的抑制，以及增加食物摄入的细胞的抑制作用。了解该细胞及其对食物摄入信号的反应，以及它调节的其他细胞应允许新的途径干预大脑如何控制体重，并最终可以增强我们扭转肥胖症流行的能力。与具有多种稳态功能的弧形核的其他神经元类似，兴奋性弧形谷氨酸神经元的功能除了能量稳态外，还包括其他角色。