AgRP Neuron Activity – Plasticity, Gene Expression and Excitatory Afferent Control

AgRP 神经元活性 — 可塑性、基因表达和兴奋性传入控制

• 批准号: 9098186
• 负责人: BRADFORD B LOWELL
• 金额: $ 62.88万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9098186
• 关键词: Afferent Neurons; Anatomy; Appetite Stimulants; Applications Grants; Area; Atlases; Biosensor; Brain; Cataloging; Catalogs; Cell Nucleus; Cells; Complex; Cues; Dendritic Spines; Drops; Energy Metabolism; Fasting; Fluorescence Resonance Energy Transfer; Food; Gene Expression; Gene Expression Profile; Glutamates; Goals; Grant; Heterogeneity; Hormones; Hunger; Hypothalamic structure; Image; Individual; Lateral; Lead; Leptin; Maps; Measures; Mediating; Methods; Molecular; Molecular Profiling; Monitor; Mus; Neurons; Obesity; Pathway interactions; Phosphotransferases; Play; Process; RNA Sequences; Rabies; Role; Site; Slice; Structure of nucleus infundibularis hypothalami; Synapses; Synaptic plasticity; Techniques; Technology; Testing; awake; base; design; energy balance; food consumption; ghrelin; in vivo; innovation; interest; novel; novel therapeutic intervention; p21 activated kinase; public health relevance; relating to nervous system; response; sensor; transcriptome sequencing

 DESCRIPTION (provided by applicant): AgRP neurons exert remarkable control over hunger. They are activated when stores are reduced, and once engaged, they induce intense hunger. Of great interest are the means by which AgRP neuron activity is controlled. While circulating hormones like leptin and ghrelin have direct effects on AgRP neurons, AgRP neurons also receive extensive neural input. This latter point has three important implications. First, changes in AgRP neuron activity in response to fasting could primarily be caused by alterations in the strength and number of afferent synapses (i.e. synaptic plasticity). Indeed, an important role for synaptic plasticity has already been established. Second, in addition to effects on plasticity, the fasted state is also likely sensed directly or indirectly by the afferent neurons themselves, with this information then being transmitted to AgRP neurons through the very same synapses. Third, cues other than those related to energy balance could also engage these afferents to bring about rapid changes in AgRP neuron activity. Of note, food-related cues, without any consumption of food, have recently been shown by others, and us, to rapidly reduce AgRP neuron activity. The existence of such rapid, "non-homeostatic" control of AgRP neurons has important implications, and is highly likely to be mediated by afferent neural input. This goal of this grant is to study mechanisms by which AgRP neuron activity is controlled. Aim 1 will focus on synaptic plasticity and determine how fasting upregulates dendritic spines and excitatory synaptic activity - an important means of control discovered during the previous cycle. In preliminary studies, we demonstrate that an AMPK → p21-activated kinase (PAK) pathway is key. We propose the following mechanism: increased Ca2+ in AgRP neurons (due to NMDAR activation, increased AgRP neuron firing and likely also ghrelin) → CaMMKβ → AMPK → p21-activated kinase (PAK) → excitatory plasticity. To test this we are using 2P imaging and a genetically encoded FRET-based sensor to image AgRP neuron AMPK activity moment-to-moment, both in brain slices and in awake behaving mice, in response to various perturbations. Aim 2 will use single neuron RNA-seq to a) create a "transcriptional atlas" of all neurons residing in the arcuate nucleus (using Drop-seq), b) assess the transcriptional signature of AgRP neurons in comparison to other ARC neurons, and also probe for transcriptional heterogeneity between subsets of AgRP neurons, c) assess the transcriptional effects of fasting and leptin on individual AgRP neuron gene expression using an innovative single neuron nuclei RNA-seq strategy designed to preserve in vivo states of gene expression, and d) develop a single neuron nuclei RNA- seq technique to transcriptionally identify rabies+ AgRP neuron afferents. Finally, Aim 3 will employ an innovative "2-synapse" rabies strategy to identify the anatomic sites/neurons that engage the orexigenic PVHglutamatergic neuron → AgRP neuron circuit discovered during the previous cycle. Our ultimate goal in this Aim is to identify the "information carried by these afferents.

 描述（由申请人提供）：AgRP神经元对饥饿有显著的控制作用。当储存减少时，它们被激活，一旦被激活，它们就会引起强烈的饥饿感。非常感兴趣的是控制AgRP神经元活动的方法。虽然循环激素如瘦素和生长激素释放肽对AgRP神经元有直接影响，但AgRP神经元也接受广泛的神经输入。后一点有三个重要的含义。首先，禁食引起的AgRP神经元活动的变化可能主要是由传入突触的强度和数量（即突触可塑性）的改变引起的。事实上，突触可塑性的重要作用已经被确定。其次，除了对可塑性的影响， 禁食状态也可能由传入神经元本身直接或间接地感知，然后通过相同的突触将该信息传递到AgRP神经元。第三，除了与能量平衡相关的线索之外，其他线索也可以使这些传入神经引起AgRP神经元活动的快速变化。值得注意的是，最近其他人和我们都证明，在不食用任何食物的情况下，与食物相关的线索会迅速减少AgRP神经元的活动。这种快速的，“非稳态”控制的AgRP神经元的存在具有重要的意义，并且很可能是由传入神经输入介导的。这项资助的目的是研究AgRP神经元活动的控制机制。目标1将集中在突触可塑性，并确定禁食如何上调树突棘和兴奋性突触活动-一个重要的控制手段，发现在以前的周期。在初步研究中，我们证明AMPK → p21激活激酶（PAK）途径是关键。我们提出了以下机制：AgRP神经元中Ca 2+增加（由于NMDAR激活，AgRP神经元放电增加，可能还有ghrelin）→ CaMMKβ → AMPK → p21激活激酶（PAK）→兴奋性可塑性。为了测试这一点，我们正在使用2 P成像和基于基因编码的FRET传感器来对AgRP神经元AMPK活动进行实时成像，无论是在大脑切片中还是在清醒行为的小鼠中，都是为了响应各种扰动。目标2将使用单个神经元RNA-seq来a）创建所有神经元的“转录图谱”， 弓状核内（使用Drop-seq），B）与其他ARC神经元相比，评估AgRP神经元的转录特征，并且还探测AgRP神经元亚群之间的转录异质性，c）使用创新的单神经元核RNA-seq策略评估禁食和瘦素对个体AgRP神经元基因表达的转录作用，所述策略被设计为保持基因表达的体内状态，和d）开发单神经元核RNA-seq技术以转录鉴定狂犬病+ AgRP神经元传入。最后，目标3将采用创新的“2-突触”狂犬病策略来识别参与前一周期发现的食欲原性PVH神经元→ AgRP神经元回路的解剖部位/神经元。我们的最终目标是识别这些传入神经所携带的信息。