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

 描述(申请人提供)：AgRP神经元对饥饿有显著的控制作用。当商店减少时，它们就会被激活，一旦被占用，它们就会引发强烈的饥饿。人们非常感兴趣的是控制AgRP神经元活动的方法。虽然瘦素和Ghrelin等循环激素对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，AgRP神经元对禁食反应的活性变化可能主要是由传入突触的强度和数量的变化(即突触可塑性)引起的。事实上，突触可塑性的重要作用已经确立。其次，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觉到禁食状态，然后这些信息通过完全相同的突触传递给AgRP神经元。第三，除了那些与能量平衡有关的提示外，其他提示也可以促使这些传入导致AgRP神经元活动的快速变化。值得注意的是，与食物相关的线索，在没有任何食物消耗的情况下，最近被其他人和我们证明，可以迅速减少AgRP神经元的活动。AgRP神经元的这种快速、“非稳态”控制的存在具有重要的意义，而且很可能是由传入神经输入所介导的。这项拨款的目的是研究AgRP神经元活动的控制机制。目标1将专注于突触的可塑性，并确定禁食如何上调树突棘和兴奋性突触活动--这是在上一个周期中发现的重要控制手段。在初步研究中，我们证明了AMPK→p21激活的蛋白激酶途径是关键。我们提出了以下机制：(由于NMDAR激活，AgRP神经元放电增加，可能还有Ghrelin)→CaMMKβ→AMPK→p21激活的激酶→兴奋性可塑性。为了测试这一点，我们正在使用2P成像和基于基因编码的FRET传感器来成像AgRP神经元AMPK活动的时刻，无论是在脑片上还是在清醒的行为中的小鼠，对各种扰动做出反应。目标2将使用单个神经元rna-seq来a)创建一份居住在所有神经元的“转录图谱”。 在弓状核中(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，并探索AgRP神经元亚群之间的转录异质性；c)使用旨在保存体内基因表达状态的创新的单神经元核RNA-seq策略，评估空腹和瘦素对单个AgRP神经元基因表达的转录影响；以及d)建立单神经元核RNA-seq技术，以转录识别狂犬病+AgRP神经元的传入。最后，Aim 3将采用创新的“2-突触”狂犬病策略来确定在前一个周期中发现的与促食欲素性PVH谷氨酸能神经元→AgRP神经元回路相联系的解剖位置/神经元。我们在这个目标上的最终目标是识别“这些传入所携带的信息”。

 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.