Neurons of the medial habenula regulate behavioral responses to nicotine in mouse

内侧缰核神经元调节小鼠对尼古丁的行为反应

基本信息

批准号：
10017027
负责人：
Christian Peters
金额：
$ 24.9万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
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：
2017-05-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10017027
关键词：
Acute Affect Affinity Agonist Amino Acids Area Award Behavior Behavioral Binding Biological Assay Biological Models Biophysics Blood Brain Calcium Cannulas Cause of Death Cell Nucleus Chloride Channels Chloride Ion Chronic Collaborations Complement Complex Cystic Fibrosis Data Dependence Dyes Epithalamic structure Exhibits Exposure to Family Fluorescence Genes Genetic Goals Habenula Hour Human Image Imaging Techniques Implant Infusion procedures Injections Intake Integral Membrane Protein Intravenous Ion Channel Ion Channel Gating Ion Channel Protein Ions Journals Knock-out Knockout Mice Lead Link Mastication Medial Mediating Membrane Mentors Microinjections Modeling Molecular Molecular Probes Motivation Mucous body substance Mus Neuromuscular Junction Neurons Nicotine Nicotine Dependence Nicotine Withdrawal Nicotinic Receptors Output Pathway interactions Pharmaceutical Preparations Pharmacology Phase Phenotype Physiological Physiology Play Postdoctoral Fellow Prevalence Property Publications Publishing RNA Interference Regulation Research Rewards Role Self Administration Slice Smoker Structure Sucrose Symptoms System Techniques Technology Testing Tissues Tobacco smoking behavior Training Translating Viral Wild Type Mouse Withdrawal Withdrawal Symptom addiction avoidance behavior behavior influence behavioral phenotyping behavioral response brain circuitry brain tissue deprivation design drug seeking behavior electronic cigarette use experience experimental study fluorescence imaging genetic analysis imaging study in vivo in vivo imaging interest interpeduncular nucleus knock-down microendoscope mouse model mutant neuronal circuitry neurophysiology neurotransmission nicotine cessation nicotine craving nicotine exposure nicotine seeking behavior nicotine use novel post-doctoral training preventable death programs receptor response reward processing transcriptome sequencing voltage

项目摘要

Project Summary/Abstract The long-term goal of my research is to determine the mechanism by which the brain responds to the presence of nicotine and leads to voluntary regulation of its intake. The ultimate purpose is to develop a better understanding of how positively and negatively rewarding effects of this drug can lead to nicotine craving or nicotine aversive behavior, and how these two opposing effects are both regulated by the habenulo- interpeduncular circuitry. The studies detailed in this proposal will focus specifically on the neurophysiology of the medial habenula and the behaviors associated with activity of neurons in that region. Nicotine addiction, fed by regular tobacco smoking or chewing, or more recently by e-cigarette use, is a leading cause of death in both the developed and developing world. Nicotine acts in the body as an extremely potent agonist of the eponymous nicotinic acetylcholine receptor family (nAChR), which are ion channel proteins with functions in neurotransmission in the brain and at neuromuscular junctions in the periphery. NAChr are ubiquitous throughout the brain, and the mechanisms by which nicotine influences behavior to produce physiological dependency are complex. A specific nucleus in the epithalamus called the medial habenula has been implicated as a locus where circulating nicotine binds directly to a specific subtype of nAChR, in which genetic mutants have been found to be upregulated in heavy smokers, to produce downstream behavioral responses regulating voluntary nicotine intake. Intriguingly, a recently identified calcium-activated chloride channel called TMEM16A (Transmembrane protein of unknown function 16A) is very highly expressed in the medial habenula but almost nowhere else in the brain, and it is likely to contribute strongly to the firing properties of mHb neurons, though the mechanism by which nicotine produces its effects on the habenula and its associated circuitry is not known. I propose to investigate the mechanism of nicotine aversion in medial habenula neurons, and to begin by studying the TMEM16A channel as a functional contributor to nicotine aversion. In the first aim pursued during the K99 phase, I will use self-administration assays with direct habenular nicotine microinjections, as well as withdrawal assays with long term exposure followed by deprivation, to examine how acute and chronic nicotine affects behavioral responses mediated in the medial habenula and how knockout of the Tmem16a gene in mice affects those effects. In the second aim, I will begin to more generally probe the medial habenula's function by implanting microendoscopes and using in vivo fluorescence imaging experiments to directly visualize neuronal activity in response to acute and chronic nicotine exposure. In the third aim, taking place following the transition to independence, I will combine the in vivo imaging and self-administration paradigms to rigorously investigate medial habenular neuron function in regulating nicotine self-administration in wild-type mice and models with established nicotine- seeking phenotypes traceable to the medial habenula. I will also probe the cellular basis of plasticity during onset of nicotine dependence using RNAseq analysis of medial habenula neurons in naïve and dependent mice. Finally, in aim 4, I will perform paired slice recordings of the medial habenula and the interpeduncular nucleus to investigate the input/output relationship of firing in the mHb, and how these are affected by focal applications of nicotine or other specific agonists, as well as by pre-exposure of the mice to chronic nicotine. Since joining the Jan lab in 2012, my research has focused specifically on the biophysics and pharmacology of the TMEM16A channel. Directly upon joining the lab, I undertook a short collaboration with Jan lab postdoc Fen Huang, Jason Rock, and others to explore the role of TMEM16A in airway mucus secretion and the ability of TMEM16A blockers to alleviate this in a cystic fibrosis model. Having a background in the structural biophysics of ion channel gating, I quickly became interested in how the TMEM16A channel is opened in response to elevation of intracellular calcium concentration (as would happen as a result of activation of a nicotine receptor, for instance), a study which resulted in a publication in the journal eLife, where I was co-first author. More recently, I performed a study to identify amino acids in TMEM16A important for chloride ion flux, and to characterize two novel inhibitory compounds with a high affinity for the TMEM16A ion pore. That study was published in PNAS in 2015. Over the remainder of my postdoctoral training, I would like to gain experience in experimental approaches to translate my expertise in the study of ion channel function into better understanding of how they contribute to the physiology of neuronal circuitry of the brain, with a specific focus on nicotine-sensitive circuitry involved in addiction and associated behavioral phenotypes. I believe the K99/R00 Pathway to Independence award is an ideal program for this goal, as it allows me to use both my specific areas of strength, and to acquire my desired training in the K99 phase to develop long term projects studying the neurophysiology of nicotine addiction and aversion in the R00 phase.

项目概要/摘要我研究的长期目标是确定大脑对存在做出反应的机制尼古丁并导致自愿调节其摄入量，最终目的是开发更好的尼古丁摄入量。了解这种药物的积极和消极的奖励作用如何导致尼古丁渴望或尼古丁厌恶行为，以及这两种相反的作用如何受到缰核的调节本提案中详细介绍的研究将特别关注脚间回路的神经生理学。内侧缰核以及与该区域神经元活动相关的行为。尼古丁成瘾是由于经常吸烟或咀嚼，或者最近通过使用电子烟而导致的。尼古丁在发达国家和发展中国家都是主要死亡原因。同名烟碱乙酰胆碱受体家族 (nAChR) 的有效激动剂，该受体是离子通道具有大脑神经传递和外周神经肌肉接头功能的蛋白质。 NAChr 在整个大脑中无处不在，尼古丁影响行为的机制产生的生理依赖性是复杂的。缰核被认为是循环尼古丁直接与特定亚型结合的位点。 nAChR，其中的基因突变体被发现在重度吸烟者中上调，产生有趣的是，最近发现了调节自愿尼古丁摄入量的下游行为反应。钙激活氯离子通道称为 TMEM16A（功能未知的跨膜蛋白 16A）在内侧缰核中表达非常高，但在大脑的其他地方几乎没有表达，并且它可能有助于尽管尼古丁产生作用的机制对 mHb 神经元的放电特性有强烈影响我建议研究尼古丁的机制。内侧缰核神经元的厌恶，并首先研究 TMEM16A 通道作为功能在 K99 阶段追求的第一个目标中，我将使用自我管理。直接缰核尼古丁显微注射测定，以及长期暴露的戒断测定其次是剥夺，以检查急性和慢性尼古丁如何影响介导的行为反应内侧缰核以及小鼠中 Tmem16a 基因的敲除如何影响这些效果在第二个目标中，我将开始通过植入显微内窥镜并使用体内荧光成像实验可直接可视化响应急性和在第三个目标中，发生在向独立过渡之后，我将结合起来。严格研究内侧缰核神经元的体内成像和自我管理范例在野生型小鼠和已建立尼古丁模型中调节尼古丁自我施用的功能寻找可追溯到内侧缰核的表型，我还将探讨可塑性的细胞基础。使用 RNAseq 分析幼稚和依赖的内侧缰核神经元的尼古丁依赖性发作最后，在目标 4 中，我将对内侧缰核和脚间进行配对切片记录。核研究 mHb 中放电的输入/输出关系，以及这些关系如何受到焦点的影响应用尼古丁或其他特定激动剂，以及使小鼠预先暴露于慢性尼古丁。自 2012 年加入 Jan 实验室以来，我的研究主要集中在生物物理学和 TMEM16A 通道的药理学加入实验室后，我立即与他们进行了短暂的合作。 Jan 实验室博士后 Fen Huang、Jason Rock 等人探索 TMEM16A 在气道粘液中的作用分泌以及 TMEM16A 阻滞剂在囊性纤维化模型中缓解这种情况的能力。在离子通道门控的结构生物物理学中，我很快对 TMEM16A 通道是如何工作的产生了兴趣响应于细胞内钙浓度的升高而打开（由于例如，尼古丁受体的激活），这项研究结果发表在《eLife》杂志上，其中我是共同第一作者，最近，我进行了一项研究，以确定 TMEM16A 中重要的氨基酸。氯离子通量，并表征两种对 TMEM16A 离子具有高亲和力的新型抑制化合物该研究于 2015 年发表在 PNAS 上。在我剩余的博士后培训中，我希望获得实验方法的经验，将我的专业知识转化为离子通道功能研究更好地了解它们如何对大脑神经回路的生理学做出贡献特别关注与成瘾和相关行为表型相关的尼古丁敏感回路。相信 K99/R00 独立之路奖是实现这一目标的理想计划，因为它允许我使用既是我的优势领域，也是我在 K99 阶段获得所需的培训以实现长期发展研究 R00 阶段尼古丁成瘾和厌恶的神经生理学的项目。