Zona incerta GABA neurons modulate energy homeostasis

未定带 GABA 神经元调节能量稳态

基本信息

批准号：
9426268
负责人：
ANTHONY N VAN DEN POL
金额：
$ 41.1万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9426268
关键词：
Address Adult Animals Appetite Stimulants Area Axon Axonal Transport Behavioral Body Weight Brain Brain region CASP3 gene Caspase Cell Nucleus Cells Cessation of life Country Coupled Data Desire for food Diabetes Mellitus Dopamine Eating Efferent Neurons Electron Microscopy Electrophysiology (science)Emotional Enterobacteria phage P1 Cre recombinase FOS gene Food Food deprivation (experimental)Foundations Genes Glutamates Health Heart Diseases Homeostasis Hypothalamic structure Injection of therapeutic agent Investigation Label Laser Scanning Confocal Microscopy Lasers Lateral Leptin Light LoxP-flanked allele Malignant Neoplasms Measures Methods Microinjections Mus Neuromodulator Neurons Neuropeptides Obesity Play Population Presynaptic Terminals Pseudorabies Rabies Rabies virus Regulation Reporter Reporter Genes Reporting Rewards Role Secondary to Signal Transduction Site Slice Stroke Structure Structure of paraventricular nucleus of thalamus Substrate Interaction Synapses Testing Thalamic structure Tracer Transgenic Mice Variant Viral Vector Weight Weight Gain Whole-Cell Recordings Work base behavior test brain cell cell type designer receptors exclusively activated by designer drugs dopaminergic neuron excitatory neuron experimental study feeding gamma-Aminobutyric Acid ghrelin immunocytochemistry in vivo inhibitory neuron interest mad itch virus novel novel strategies optogenetics patch clamp postsynaptic postsynaptic neurons promoter reduced food intake response trend ventromedial hypothalamic nucleus voltage clamp zona incerta

项目摘要

Title: Zona incerta GABA neurons modulate energy homeostasis Abstract. Obesity, which often leads to secondary health complications including heart disease, diabetes, stroke, cancer, and early death, has become a major health concern in the US. Here we test the general hypothesis that neurons in the rostromedial zona incerta (ZI), and particularly inhibitory GABA neurons, play an unexpectedly profound orexigenic role in increasing food intake and body weight. Most of the work done on the neuronal regulation of energy homeostasis has previously focused on neurons in other brain regions. The first Aim examines the structural substrates for interaction between ZI GABA axons and their postsynaptic targets. Using confocal scanning laser microscopy and dual immunolabel electron microscopy coupled with cre recombinase-dependent AAV and rabies virus tracers, we test the hypothesis that ZI axons project to a number of sites, including the paraventricular thalamus (PVT) and hypothalamic ventromedial nucleus (VMH) where direct synaptic connections are made with excitatory neurons. We use multiple transgenic mouse lines expressing Cre recombinase under control of various neuron-selective promoters including mice that express Cre in GABA neurons driven by a vGAT promoter. These will be coupled with intracerebral microinjections of AAV viral vectors containing floxed GFP or tdTomato reporter genes to study ZI GABA neuron efferent and afferent axon projections. We will also employ injection of a Cre recombinase-dependent Brainbow-type pseudorabies virus into the ZI; after retrograde axonal transport this PRV expresses a red reporter in wild-type cells, but in Cre-expressing GABA cells, reporter expression changes to yellow or cyan, helping define the cell of interest. Aim 2 tests the hypothesis that ZI GABA cells respond to long distance signals of energy homeostasis including ghrelin and leptin, and also to axonally released neuropeptide modulators of food intake. Whole cell recording allows us to test different mechanisms of action on ZI GABA cells produced by neuromodulator signals from other neurons involved in energy homeostasis. C-fos expression will be examined after food deprivation to test the hypothesis that ZI GABA neurons are more active during reduced food availability. Optogenetics is used in brain slices to test the hypothesis that release of transmitter from ZI GABA axons will exert similar inhibitory effects on PVT and VMH neurons; neighboring ZI dopamine cells are also tested. In Aim 3, we examine the role that rostromedial ZI GABA neurons play in ongoing energy homeostasis by cell silencing (using Gi-DREADDs and caspase) to test the hypothesis that body weight and food intake is reduced. Optogenetic activation with ChR2 variant ChIEF will test the hypothesis that stimulation of ZI GABA axons in different terminal zones will each enhance food intake and body weight. We also test the hypothesis that ZI GABA neuron activation provides a positive emotional valence. Together, these experiments examine converging structural, electrophysiological, and behavioral analyses, focusing on the role of the GABA ZI neurons in energy homeostasis. With the growing levels of obesity in this country approaching 30% of the adult population, and the associated health complications, identifying and understanding the brain cells that control and sense energy homeostasis will help to identify novel approaches to reducing the trend toward obesity.

标题：不确定带 GABA 神经元调节能量稳态抽象的。肥胖通常会导致继发性健康并发症，包括心脏病、糖尿病、中风、癌症和过早死亡已成为美国的主要健康问题。这里我们测试一下一般情况假设嘴内侧未定带 (ZI) 中的神经元，特别是抑制性 GABA 神经元，发挥作用在增加食物摄入量和体重方面具有意想不到的深远的促食欲作用。大部分工作已完成关于能量稳态的神经元调节以前主要集中在其他大脑区域的神经元上。第一个目标是检查 ZI GABA 轴突与其相互作用的结构底物突触后目标。使用共焦扫描激光显微镜和双免疫标记电子显微镜结合 cre 重组酶依赖性 AAV 和狂犬病病毒示踪剂，我们检验了 ZI 的假设轴突投射到许多部位，包括室旁丘脑 (PVT) 和下丘脑腹内侧核（VMH）与兴奋性神经元建立直接突触连接。我们使用在各种神经元选择性控制下表达 Cre 重组酶的多个转基因小鼠系启动子，包括在 vGAT 启动子驱动的 GABA 神经元中表达 Cre 的小鼠。这些将是结合脑内显微注射含有 floxed GFP 或 tdTomato 报告基因的 AAV 病毒载体研究 ZI GABA 神经元传出和传入轴突投射的基因。我们还将采用 Cre 注射重组酶依赖性 Brainbow 型伪狂犬病病毒进入 ZI；逆行轴突运输后 PRV 在野生型细胞中表达红色报告基因，但在表达 Cre 的 GABA 细胞中，报告基因表达变为黄色或青色，帮助定义感兴趣的细胞。目标 2 检验 ZI GABA 细胞的假设对能量稳态的长距离信号（包括生长素释放肽和瘦素）以及轴突作出反应释放食物摄入的神经肽调节剂。全细胞记录使我们能够测试不同的来自其他神经元的神经调节信号产生的 ZI GABA 细胞的作用机制能量稳态。食物剥夺后将检查 C-fos 表达，以检验 ZI 的假设 GABA 神经元在食物供应减少时更加活跃。光遗传学用于脑切片测试 ZI GABA 轴突释放递质将对 PVT 产生类似的抑制作用的假设 VMH 神经元；邻近的 ZI 多巴胺细胞也进行了测试。在目标 3 中，我们研究了以下角色：嘴内侧 ZI GABA 神经元通过细胞沉默来维持持续的能量稳态（使用 Gi-DREADD）和半胱天冬酶）来检验体重和食物摄入量减少的假设。光遗传学激活 ChR2 变体 ChIEF 将检验以下假设：刺激不同末端区域的 ZI GABA 轴突将每种都可以增加食物摄入量和体重。我们还测试了 ZI GABA 神经元激活的假设提供积极的情绪效价。这些实验共同检验了聚合结构、电生理学和行为分析，重点关注 GABA ZI 神经元在能量中的作用体内平衡。随着这个国家肥胖率不断上升，接近成年人口的 30%，并且相关的健康并发症，识别和理解控制和感知的脑细胞能量稳态将有助于找到减少肥胖趋势的新方法。