Competing effects of AgRP and POMC neurons on cAMP signaling in downstream neurons in vivo

项目总结 下丘脑AgRP饥饿神经元和POMC饱腹神经元使用神经肽AgRP、NPY和αMSH来 调节食物摄入量、能量消耗和体重。一个重要的下游目标是满足感- 促进下丘脑室旁核MC4R受体表达神经元(PVHMC4R)； 它们从AgRP和POMC神经元接收汇聚的肽能信号。然而，PVHMC4R到底是如何 神经元解释和整合这些肽信号仍然不清楚，特别是关于参与 下游的第二个信使营地。核心未知数可以归结为三个问题： (I)饥饿和饱腹肽如何改变PVHMC4R神经元中的cAMP？ (Ii)cAMP如何调节PVHMC4R神经元的放电？ (Iii)在PVHMC4R神经元中，cAMP信号对调节PVHMC4R活性的影响最大？ 回答这些问题的主要障碍是缺乏工具来测量和操纵PVHMC4R中的cAMP 神经元。最近，我通过帮助开发和应用分子和光学工具包来克服这个障碍 在行为过程中测量、建立和降级营地。这所学校的学习和职业发展活动 K99/R00提案旨在为我提供启动独立研究所需的培训 该项目应用新的cAMP工具来了解PVHMC4R神经元中的肽信号。我假设 AgRP和POMC神经元活动的协调相反变化调节PVHMC4R神经元的峰电位 通过营地信号上的竞争行动。为了验证这一假设，我提出了以下目标： 目的1：用光学传感器Caddis对PVHMC4R神经元的cAMP动态进行实验成像 诱导和摄食诱导的多肽释放，并比较禁食和摄食状态下的cAMP信号。 目的2：测量如何直接增加cAMP(使用一种新的光遗传工具，BIPAC)和降解cAMP (使用工程磷酸二酯酶，PDE4D3-Cat)改变PVHMC4R神经元的可塑性和尖峰。 目的3：设计和使用亚细胞靶向的Caddis、BIPAC、PDE4D3-Cat，以了解当地 PVHMC4R神经元中的cAMP信号。 拟议的研究将由Mark Andermann博士和Bradford Lowell博士指导，他们还将提供 关于成为独立研究人员的指导意见。我将学习脑室内给药的新技术， 全细胞膜片钳，以及我的导师和亚历克斯·班克斯博士对能量消耗的测量， 玛丽亚·莱蒂宁和约瑟夫·马祖布。我将接受高士强博士的蛋白质工程培训。 内格尔、贝尔纳多·萨巴蒂尼和加里·耶伦。我将从上述专家和课程中获得新的技能 和科学会议，结合我在营地研究方面的现有专业知识。这将使我能够 了解何时、何地以及如何使用cAMP来滴定PVHMC4R神经元的活性。这些结果将填补一个 在开发和了解肥胖症治疗方法方面的重要知识差距。

PROJECT SUMMARY Hypothalamic AgRP hunger neurons and POMC satiety neurons use neuropeptides AgRP, NPY, and αMSH to regulate food intake, energy expenditure, and body weight. One important downstream target is the satiety- promoting MC4R receptor-expressing neurons in the paraventricular nucleus of the hypothalamus (PVHMC4R), which receive converging peptidergic signals from AgRP and POMC neurons. However, exactly how PVHMC4R neurons interpret and integrate these peptide signals remains unclear, especially regarding the involvement of the downstream second messenger cAMP. The central unknowns can be summarized into three questions: (i) How do hunger and satiety peptides alter cAMP in PVHMC4R neurons? (ii) How does cAMP regulate the spiking of PVHMC4R neurons? (iii) Where in a PVHMC4R neuron is cAMP signaling most influential in regulating PVHMC4R activity? The main barrier to answering these questions is the lack of tools to measure and manipulate cAMP in PVHMC4R neurons. I have recently overcome this barrier by helping develop and apply a molecular and optical toolset to measure, make, and degrade cAMP during behaviors. The studies and career development activity in this K99/R00 proposal are designed to provide me with the necessary training to initiate an independent research program that applies the new cAMP tools to understand peptide signaling in PVHMC4R neurons. I hypothesize that coordinated, opposing changes in AgRP and POMC neuron activity regulate spiking in PVHMC4R neurons via competing actions on cAMP signaling. To test this hypothesis, I propose the following aims: Aim 1: To image cAMP dynamics in PVHMC4R neurons with the optical sensor cADDis during experimentally induced and feeding-induced peptide release, and to compare cAMP signaling between fasted and fed states. Aim 2: To measure how directly increasing cAMP (using a new optogenetic tool, biPAC) and degrading cAMP (using an engineered phosphodiesterase, PDE4D3-Cat) changes plasticity and spiking of PVHMC4R neurons. Aim 3: To engineer and use subcellularly targeted cADDis, biPAC, PDE4D3-Cat in order to understand local cAMP signaling in PVHMC4R neurons. The proposed research will be mentored by Drs. Mark Andermann and Bradford Lowell, who will also provide guidance on becoming an independent researcher. I will learn new techniques in intraventricular drug delivery, whole-cell patch-clamp, and measurements of energy expenditure from my mentors as well as Drs. Alex Banks, Maria Lehtinen, and Joseph Majzoub. I will receive training in protein engineering from Drs. Shiqiang Gao, Georg Nagel, Bernardo Sabatini, and Gary Yellen. I will acquire new skills from the above experts and from courses and scientific meetings, combined with my existing expertise in cAMP research. This will enable me to understand when, where, and how cAMP is used to titrate PVHMC4R neurons activity. These results will fill an important knowledge gap in developing and understanding treatments for obesity.