Arcuate nucleus glutamatergic neurons modulate 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日益流行，现在影响着30%的成年人口，并由此导致心脏病、高血压、糖尿病、关节功能障碍、中风、癌症和早期死亡的增加，据估计，每年的成本超过750亿美元。许多因素导致了今天的肥胖问题。下丘脑弓状核是大脑能量平衡的信息中枢，既接收来自参与能量储存或释放的外周器官的信息，也接收来自同样在中枢神经系统调节能量稳态中起重要作用的大脑其他区域的轴突信息，并发出调节食物摄入和利用的输出信息。多年来，这个领域的一个焦点一直是参与能量调节的神经肽，以及分泌它们的下丘脑神经元。大多数参与调节能量稳态的关键肽都与弓形核中的抑制性递质GABA共定位。这个应用程序的重点是我们已经确定的在中枢神经系统调节能量平衡的新细胞参与者，弓形谷氨酸能神经元，一个具有减少食物摄入特征的细胞。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，对谷氨酸神经元的关注相对较少，尽管事实上在谷氨酸受体拮抗剂的存在下，在弓形核或下丘脑的其他地方几乎没有兴奋性突触活动。在提交此申请之前，我们已经解决了一个中心问题，即识别这些与其他下丘脑细胞没有形态差异的谷氨酸细胞，通过在谷氨酸神经元中产生一个转基因小鼠，该转基因小鼠在泡状谷氨酸转运蛋白2 （vGluT2）的控制下选择性地表达报告基因GFP。我们的实验利用了全细胞膜片钳电生理学、氟金和伪狂犬病毒的通道示踪、超微结构免疫细胞化学和全动物能量平衡挑战背景下改变的基因表达的组合。第一组实验解决了谷氨酸神经元表现出与弓形核的抑制性神经元相同的传出轴突投射的假设。这将用氟金和重组伪狂犬病毒显微注射到假定的目标区域进行测试。为了验证弓形形谷氨酸细胞调节厌氧原皮质素（POMC）神经元活性的假说，我们将在POMC神经元上记录用兴奋性微滴法刺激局部谷氨酸细胞激活细胞体而非传代轴突的情况。平行实验解决了谷氨酸细胞是否相互支配，从而增加其输出的时间和功率的问题。超微结构双标记免疫细胞化学将用于验证局部摄氧神经肽Y （NPY）免疫反应轴突与谷氨酸细胞突触接触的假设，类似于与厌氧性POMC神经元突触的NPY轴突。第二组实验，使用全细胞膜片钳记录下丘脑切片，解决了“是什么活性或被动膜特征使谷氨酸神经元独特”的问题，与弓形核的gaba能神经元相比，已经得到了大量关注。第三组电生理实验验证了从其他弓状核神经元释放的参与调节能量稳态的神经肽调节弓状谷氨酸神经元活动的假设。在第四组实验中，我们询问弓形谷氨酸神经元是否对与能量稳态有关的远距离信号作出反应，特别是葡萄糖和瘦素。总之，这些实验将揭示弓形核中一个独特的、以前未被表征的兴奋性神经元的组织和细胞行为和反应。了解这些谷氨酸能细胞可以让我们更好地了解能量稳态和体重调节的细胞机制，并可以让我们对通过控制食物摄入和消耗的神经元治疗肥胖的潜在方法有新的认识。弓状核中的许多神经元具有多种功能；谷氨酸细胞可能也不例外，它可能在大脑这个小而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、代谢和应激反应。

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. 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.