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

项目总结/摘要 我研究的长期目标是确定大脑对存在的反应机制 尼古丁，并导致其摄入量的自愿调节。最终目的是为了更好地发展 了解这种药物的积极和消极奖励作用如何导致尼古丁渴望， 尼古丁厌恶行为，以及这两种相反的作用是如何由缰核调节的- 脚间回路本提案中详细介绍的研究将特别关注以下神经生理学： 内侧缰和与该区域神经元活动相关的行为。 尼古丁成瘾，通过定期吸烟或咀嚼烟草，或最近使用电子烟，是一种 是发达国家和发展中国家的主要死因。尼古丁在体内的作用是一种 表型烟碱乙酰胆碱受体家族（nAChR）的强效激动剂， 在大脑和外周神经肌肉接头的神经传递中起作用的蛋白质。 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。上丘脑的一个特殊的核团叫做内侧核团 缰被认为是循环尼古丁直接结合特定亚型的位点， 在重度吸烟者中发现基因突变体上调的nAChR， 调节尼古丁自愿摄入的下游行为反应。有趣的是，最近发现的一种 钙激活的氯离子通道称为TMEM 16 A（功能未知的跨膜蛋白16 A）， 在内侧缰核中表达非常高，但在大脑的其他地方几乎没有，它可能有助于 强烈的mHb神经元的放电特性，虽然尼古丁产生其影响的机制， 缰及其相关的神经回路的情况尚不清楚。我建议研究尼古丁的作用机制 厌恶内侧缰神经元，并开始通过研究TMEM 16 A通道作为一个功能性的 对尼古丁的厌恶。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，以及长期暴露的戒断试验 然后剥夺，以检查急性和慢性尼古丁如何影响行为反应介导的， 内侧缰核以及敲除小鼠中的Tmem 16 a基因如何影响这些效应。第二个目标， 我将开始通过植入显微内窥镜和使用 在体内荧光成像实验中， 慢性尼古丁暴露第三个目标是在过渡到独立之后实现，我将把联合收割机 严格研究内侧缰核神经元体内成像和自我给药模式 在野生型小鼠和已建立的尼古丁- 寻找可追溯到内侧缰的表型我还将探索可塑性的细胞基础， 使用RNAseq分析未经处理和依赖的内侧缰核神经元的尼古丁依赖发作 小鼠最后，在目标4中，我将进行内侧缰和脚间的成对切片记录 研究mHb中放电的输入/输出关系，以及这些如何受到焦点的影响。 尼古丁或其他特定激动剂的应用，以及通过将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 TMEM 16 A通道的药理学。刚加入实验室，我就与 Jan实验室博士后Fen Huang，Jason Rock等人探索TMEM 16 A在气道粘液中的作用 在囊性纤维化模型中，TMEM 16 A的分泌和TMEM 16 A阻断剂减轻这种分泌的能力。具有背景 在离子通道门控的结构生物物理学中，我很快对TMEM 16 A通道是如何 响应于细胞内钙浓度的升高而打开（如由于 例如，尼古丁受体的激活），这项研究在eLife杂志上发表， 我是第一作者。最近，我进行了一项研究，以确定TMEM 16 A中的氨基酸对 氯离子通量，并表征两种新型的抑制性化合物与TMEM 16 A离子的高亲和力 毛孔该研究于2015年发表在PNAS上。在我的博士后培训的剩余时间里，我想 获得实验方法方面的经验，将我在离子通道功能研究方面的专业知识转化为 为了更好地了解它们如何有助于大脑神经元回路的生理学， 特别关注尼古丁敏感回路参与成瘾和相关的行为表型。我 我相信K99/R 00独立之路奖是实现这一目标的理想计划，因为它使我能够使用 这两个我的具体领域的优势，并获得我想要的培训，在K99阶段，以发展长期 研究R 00阶段尼古丁成瘾和厌恶的神经生理学项目。

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.