Arcuate Nucleus Glutamatergic Neurons Modulate Energy Homeostasis

描述（由申请人提供）：本申请所解决的潜在健康问题是肥胖症的日益流行，其现在影响30%的成年人口，以及由此导致的心脏病、高血压、糖尿病、关节功能障碍、中风、癌症和过早死亡的增加，估计每年花费超过750亿美元。许多因素导致了今天的肥胖问题。下丘脑弓状核是能量平衡的信息中枢，既接受来自周围器官的能量储存和释放信息，又接受来自其他脑区的轴突信息，这些轴突信息在中枢神经系统能量平衡的调节中起重要作用，并发出传出信息调节食物的摄入和利用。多年来，该领域的一个焦点是参与能量调节的神经肽和分泌它们的下丘脑神经元。大多数参与能量稳态调节的关键肽与弓状核中的抑制性递质GABA共定位。这个应用程序的重点是什么似乎是一个新的细胞球员在中枢神经系统调节能量平衡，我们已经确定，弓状神经元，一个细胞，有一个，减少食物摄入量的轮廓。在大脑的大多数其他区域，谷氨酸被认为是一种主要的神经递质。但在下丘脑，相对较少的注意已经给予谷氨酸神经元，尽管事实上，在谷氨酸受体拮抗剂的存在下，几乎没有兴奋性突触活动在弓状核，或在下丘脑的其他地方。在提交本申请之前，我们已经解决了一个核心问题，即通过产生在囊泡谷氨酸转运蛋白2（vGluT 2）的控制下选择性地在谷氨酸神经元中表达报告GFP的转基因小鼠，识别这些与其他下丘脑细胞没有形态学差异的谷氨酸细胞。我们的实验利用全细胞膜片钳电生理学，荧光金和伪狂犬病病毒，超微结构免疫细胞化学，并在整个动物能量平衡的挑战的背景下改变基因表达的跟踪相结合。第一组实验解决的假设，谷氨酸神经元显示出相同的传出轴突的弓状核的抑制性神经元的预测。这将用荧光金和重组伪狂犬病病毒显微注射到假定的靶区域进行测试。为了验证弓状谷氨酸细胞调节促凋亡的阿黑皮素原（proopiomelanocortin，POMC）神经元活性的假设，我们将记录POMC神经元的活动，同时用兴奋性微滴法刺激局部谷氨酸细胞，激活胞体而不是通道轴突。平行实验解决了谷氨酸细胞是否相互支配的问题，从而增加了它们输出的时间和功率。超微结构的双标记免疫细胞化学将被用来测试的假设，当地orexigenic神经肽Y（NPY）免疫反应性轴突与谷氨酸细胞突触接触，类似的神经肽Y轴突与orexigenic POMC神经元突触。第二组实验，使用全细胞膜片钳记录在下丘脑切片，解决的问题是“什么主动或被动的膜特性使谷氨酸神经元独特的”，相比，GABA能神经元的弓状核，已收到实质性的关注。第三组电生理学实验测试的假设，从其他弓状核神经元参与调节能量稳态释放的神经肽调节活动的弓状谷氨酸神经元。在第四组实验中，我们询问弓状谷氨酸神经元是否对与能量稳态有关的长距离线索做出反应，特别是葡萄糖和瘦素。总之，这些实验将揭示弓状核中一个独特的、以前未被表征的兴奋性神经元的组织、细胞行为和反应。了解这些神经元能细胞应该让我们更好地理解能量稳态和体重调节的细胞机制，并应该让我们对通过控制食物摄入和支出的神经元治疗肥胖症的潜在治疗有新的认识。弓状核中的许多神经元有多种作用;谷氨酸细胞可能也不例外，并且可能在大脑的这个小但关键的部分控制的其他功能中发挥作用，包括调节垂体和其他内分泌器官，生殖和哺乳，生长，代谢和对压力的反应。

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.