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（由适用提供）：AGRP神经元对饥饿产生明显的控制。当商店减少并且一旦参与，它们会影响强烈的饥饿时，它们会被激活。引人注目的是控制AGRP神经元活动的手段。虽然循环激素（如瘦素和生长素蛋白）对AGRP神经元具有直接影响，但AGRP神经元也接受了广泛的神经元输入。这一点有三个重要的含义。首先，AGRP神经活动对禁食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。确实，已经确立了突触可塑性的重要作用。第二，除了对可塑性的影响外， 传入神经元本身也可能直接或间接地感知禁食状态，然后通过相同的突触将此信息传输到AGRP神经元。第三，除了与能量平衡相关的提示外，其他提示也可以吸引这些传入，从而导致AGRP神经元活动的快速变化。值得注意的是，与食物相关的提示，没有任何食物消费，而其他人也表明了我们迅速降低AGRP神经元活性。对AGRP神经元的这种快速，“态度”控制的存在具有重要意义，并且很可能是由传入的神经元输入介导的。这项赠款的这个目标是研究控制AGRP神经元活动的机制。 AIM 1将重点放在合成可塑性上，并确定禁食的上调树突状刺和兴奋性合成活性 - 在上一个周期中发现的一种重要控制手段。在初步研究中，我们证明了AMPK→P21激活的激酶（PAK）途径是关键。我们提出了以下机制：AGRP神经元中的Ca2+增加（由于NMDAR激活，AGRP神经元发射增加，可能还增加了Ghrelin）→CAMMKβ→AMPK→AMPK→P21激活激酶（PAK）→兴奋性可塑性。为了测试这一点，我们使用2P成像和一般编码的基于FRET的传感器来对AGRP神经元AMPK活性矩到臂的影响，无论是在大脑切片还是在醒着的行为行为小鼠中，以响应各种扰动。 AIM 2将使用单个神经元RNA-seq到A）创建所有居住的神经元的“转录地图集” in the arcuate nuclearus (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基因表达状态和d）开发单个神经元核RNA-SEQ技术，以转录识别狂犬病+ AGRP神经元传入。最后，AIM 3将采用创新的“ 2-伴随”狂犬病策略来识别与前一个周期中发现的Orexenic Pvhglutamatergic神经元→AGRP神经元电路的解剖部位/神经元。在此目标中，我们的最终目标是确定“这些传入的信息。