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

项目摘要/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 尼古丁的摄入量，并导致自愿调节其摄入量。最终目的是发展一个更好的 了解这种药物的积极和消极回报效应如何导致尼古丁渴望或 尼古丁厌恶行为，以及这两种相反的影响如何都受到Habenulo- 脚间回路。这项建议中详细说明的研究将特别集中在脑血管疾病的神经生理学方面。 内侧缰核及其与该区域神经元活动相关的行为。 尼古丁成瘾，由经常吸烟或咀嚼，或最近通过使用电子烟助长，是一种 在发达国家和发展中国家都是主要死因。尼古丁在体内的作用是一种极端的 同名烟碱型乙酰胆碱受体家族(NAChR)的有效激动剂，它们是离子通道 在大脑和外周的神经肌肉连接处具有神经传递功能的蛋白质。 NAChr在大脑中无处不在，尼古丁影响行为的机制 产生生理依赖是复杂的。上皮层中一种特殊的核，称为内侧核 缰核被认为是一个基因座，循环中的尼古丁直接与特定亚型的 NAChR，其中基因突变已被发现在重度吸烟者中上调，以产生 调节自愿尼古丁摄入量的下游行为反应。有趣的是，最近发现了一种 钙激活的氯离子通道称为TMEM16A(功能未知的跨膜蛋白16A)是 它在内侧缰核高度表达，但在大脑的其他部位几乎没有表达，它可能对 对MHB神经元的放电特性有强烈的影响，尽管尼古丁产生其作用的机制 缰核及其相关回路上的情况尚不清楚。我建议研究尼古丁的作用机制 内侧缰核神经元的厌恶，并从TMEM16A通道的功能研究开始 尼古丁厌恶的推动者。在K99阶段追求的第一个目标中，我将使用自我管理 直接缰核尼古丁微量注射试验和长期暴露的戒断试验 然后是剥夺，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TMEM16A基因敲除对这些影响的影响。在第二个目标中， 我将开始通过植入显微内窥镜和使用 在体荧光成像实验，以直接可视化神经元的活动对急性和 长期接触尼古丁。在第三个目标中，在向独立过渡之后，我将结合 严谨研究内侧缰核神经元的活体成像和自我给药范式 在野生型小鼠和已建立的尼古丁模型中调节尼古丁自我给药的作用- 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 用幼稚和依赖的内侧缰核神经元的RNAseq分析尼古丁依赖的开始 老鼠。最后，在目标4中，我将对内侧缰核和脚间进行成对的切片记录 研究MHB中放电的输入/输出关系，以及焦点如何影响这些输入/输出关系 尼古丁或其他特定激动剂的应用，以及将小鼠预先暴露于慢性尼古丁。 自从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。一加入实验室，我就与 Jan Lab博士后黄芬、Jason Rock等人探索TMEM16A在呼吸道粘液中的作用 在囊性纤维化模型中，TMEM16A阻滞剂的分泌和减轻这种情况的能力。有背景的 在离子通道门控的结构生物物理学中，我很快对TMEM16A通道是怎样的感兴趣 响应细胞内钙浓度的升高而打开(由于 例如，尼古丁受体的激活)，这项研究导致在eLife杂志上发表了一篇文章，其中 我是第一作者之一。最近，我进行了一项研究，以确定TMEM16A中对 氯离子通量，并表征了两种新的对TMEM16A离子具有高亲和力的抑制化合物 毛孔。该研究于2015年发表在《美国国家科学院院刊》上。在我剩余的博士后培训中，我希望 在实验方法方面获得经验，将我在离子通道功能研究方面的专业知识转化为 为了更好地理解它们如何对大脑神经回路的生理做出贡献， 特别关注与成瘾有关的尼古丁敏感回路和相关的行为表型。我 我相信K99/R00独立之路奖是实现这一目标的理想方案，因为它允许我使用 无论是在我的特长领域，还是在K99阶段获得我想要的训练，都能长期发展 研究R00阶段尼古丁成瘾和厌恶的神经生理学的项目。

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.