The Habenulo-Interpeduncular Pathway and Nicotine

描述（由申请人提供）：烟草中含有的尼古丁被认为是导致人类吸烟者烟草成瘾的主要强化成分。尼古丁的积极作用涉及大脑的中边缘奖励通路，而其负面作用似乎是由habenuol - interduncular通路介导的。确定调节消耗尼古丁的动机信号的机制对于理解导致人类吸烟者烟草成瘾的成分至关重要。PI过去的经验已经确定了habenuhi - interpedle通路在抑制高剂量尼古丁摄入方面的功能。通过本研究，PI建立并验证了小鼠尼古丁自我给药模型，学习了组织中基因分型和mRNA表达评估的分子技术，协助慢病毒载体的开发和验证，为提出的研究提供了基础。本研究的近期目标有两个：(1)进一步明确habenulo- interponducular pathway及其传入/传出调节抑制动机信号以限制尼古丁消耗的机制；(2)为PI的职业和专业发展提供必要的培训经验，以确保PI作为独立学术科学家的成功。关于第一个目标，PI将应用小鼠体内神经光遗传学技术来更清楚地定义参与抑制动机信号的通路，神经递质和受体的具体作用。将光激活的抑制性盐紫红质泵（eNpHR3.0）注射到选定的脑区，以便在BAC转基因胆碱乙酰转移酶（ChAT）： Cre小鼠中转染胆碱能神经元/轴突。随后，该泵的光介导激活对静脉尼古丁自我给药行为的影响将被检查。值得注意的是，这一建议是高度创新的，它结合了两种新技术——小鼠静脉注射尼古丁自我给药和光遗传学——来定义调节尼古丁行为的神经元过程。PI将得到Garret Stuber博士、Peter Kalivas博士和David Fitzpatrick博士的进一步协助/培训。神经递质和受体参与信号处理和蛋白质的变化与急性和慢性尼古丁自我给药也将被检查。本研究的总体假设是后隔将抑制性动机信号传递到内侧束（MHb），然后将该信号传递到脚间核（IPN），再传递到背侧被盖核（DTN），以抑制尼古丁的行为自我给药。进一步假设表达？5个烟碱乙酰胆碱受体（nAChR）亚基调节IPN-DTN通路内的信息处理，该信息随后从DTN传递到腹侧被盖区。本提案中概述的实验将检验这些假设，并可能因此为人类吸烟者尼古丁习惯的潜在机制提供有价值的见解。此外，斯克里普斯研究所的良好环境将为完成K99阶段的研究提供必要的设备、空间、设施和科学支持。通过这种训练，PI可能会开始解决长期目标，即阐明尼古丁调节大脑功能的机制，以及神经元活动改变行为以促进或抑制进一步吸食尼古丁的机制。因此，独立阶段的R01批准申请可能会关注MHb-IPN通路中的其他nAChR亚基和信号机制（多巴胺、血清素、脑啡肽等），因为它们参与尼古丁强化、依赖和戒断。这些发现可能为进一步确定新的药物靶点提供基础，以开发更有效的戒烟疗法。关于职业发展，本建议中有指导的职业培训对于确保私家侦探今后作为一名独立研究调查员取得成功同样至关重要。首先，PI将通过参加冷泉港或伍兹霍尔的相关课程获得教学指导，并将与导师和/或咨询委员会成员讨论的文献进行深入阅读。第二，项目负责人将接受实验室管理和拨款准备与管理方面的培训。除了撰写一份完整而透彻的R01奖助金提案外，这次培训还将提高编制预算、跟踪支出、保持方向和编制进度报告的知识。进一步的指导将通过参加由NIH校外研究办公室提供的NIH项目资金和资助管理区域研讨会获得。第三，与导师和新成立的咨询委员会的互动将提供有关进展和演讲技巧的必要反馈，以及与求职和向独立过渡相关的输入。这些人在药物成瘾领域拥有丰富的知识，拥有大量的R01和其他资助，在NIH和非NIH资助审查委员会任职，担任期刊编辑，并参与教师招聘。此外，PI将参加由佛罗里达斯克里普斯大学职业和博士后服务办公室和科学女性网络提供的职业发展研讨会，并将参加负责任的研究行为课程。因此，通过实际操作，教学和指导培训，该计划将大大提高PI在新技术（光遗传学）方面的研究技能，使她能够在新颖，创新的条件下使用该技术（与小鼠自我管理相结合），并提高她的专业知识，以确保成功和科学独立性，在独立的早期获得R01资助作为终身教职员工。

DESCRIPTION (provided by applicant): Nicotine contained in tobacco is considered to be the primary reinforcing component responsible for tobacco addiction in human smokers. The positive effects of nicotine involve the brain's mesolimbic reward pathways whereas the aversive effects appear to be mediated by the habenulo-interpeduncular pathway. Defining the mechanisms that regulate the motivational signals to consume nicotine is essential to understanding the components that contribute to tobacco addiction in human smokers. The PI's past experiences have defined the function of the habenulo-interpeduncular pathway with respect to inhibition of intake at higher doses of nicotine. Through this research, the PI has developed and validated a mouse model for nicotine self-administration, learned molecular techniques for genotyping and assessing mRNA expression in tissue, and assisted with the development and validation of lentiviral vectors, all of which provide a basis for the research proposed. The immediate goals of this proposal are twofold: (1) to further define the mechanisms of the habenulo-interpeduncular pathway and its afferents/efferents that regulate an inhibitory motivational signal to limit nicotine consumption, and (2) to provide an essential training experience for career and professional development to ensure success of the PI as an independent academic scientist. With regard to the first goal, the PI will apply the in vivo mouse neurooptogenetic technique to more clearly define the specific role of the pathways, neurotransmitters and receptors involved in an inhibitory motivational signal. The light-activated inhibitory halorhodopsin pump (eNpHR3.0) will be injected into select brain regions to permit transfection of cholinergic neurons/axons in BAC transgenic choline acetyltransferase (ChAT): Cre mice. Subsequently, the effect of light-mediated activation of this pump on intravenous nicotine self-administration behavior will be examined. It should be noted that this proposal is highly innovative by combining two novel techniques - mouse intravenous nicotine self-administration and optogenetics - to define the neuronal processes regulating nicotine's actions. The PI will receive further assistance/training from Dr. Garret Stuber, Dr. Peter Kalivas, and Dr. David Fitzpatrick. Neurotransmitter and receptor involvement in signal processing and protein changes with acute and chronic nicotine self-administration will also be examined. The overall hypothesis of this study is that the posterior septum transmits an inhibitory motivational signal t the medial habenula (MHb) and that this signal is then relayed to the interpeduncular nucleus (IPN) and then to the dorsal tegmental nucleus (DTN) to inhibit behavioral self-administration of nicotine. It is further hypothesized that cholinergic neurons expressing the ?5 nicotinic acetylcholine receptor (nAChR) subunit modulate information processing within the IPN-DTN pathway and that information is subsequently relayed from the DTN to the ventral tegmental area. Experiments outlined in this proposal will test these hypotheses and may thereby provide valuable insights into the mechanisms underlying the nicotine habit in human tobacco smokers. Further, the excellent environment at The Scripps Research Institute will provide the equipment, space, facilities, and scientific support necessary to complete the studies for the K99 phase. With this training, the PI may then begin to address long-term goals of elucidating the mechanisms by which nicotine modulates brain function and by which neuronal activity alters behavior to promote or inhibit further consumption of the drug. As such, R01 grant submissions during the independent phase may focus on other nAChR subunits and signaling mechanisms (dopamine, serotonin, enkephalin, etc.) in the MHb-IPN pathway for their involvement in nicotine reinforcement, dependence and withdrawal. These findings may then provide a basis for further identification of novel drug targets with the goal of developing more efficacious smoking cessation therapeutics. With regard to career development, the mentored career training in this proposal will be equally essential to ensure the PI's future success as an independent research investigator. First, the PI will obtain didactic instruction by attending a relevant course at eithr Cold Spring Harbor or Woods Hole and will conduct intensive reading of the literature to be discussed with the mentor and/or advisory committee members. Second, the PI will be trained in laboratory management and grant preparation and management. In addition to writing a complete and thorough R01 grant proposal, this training will promote knowledge of the preparation of budgets, tracking of expenditures, maintaining direction and preparation of progress reports. Further instruction will be obtained by attending an NIH Regional Seminar on Program Funding and Grants Administration offered by the NIH Office of Extramural Research. Third, the interactions with the mentor and newly-formed advisory committee will provide necessary feedback regarding progress and presentation skills, as well as input relative to job searches and transitioning to independence. These individuals have a great wealth of knowledge of the drug addiction field, hold numerous R01 and other grants, serve on NIH and non-NIH grant review committees, serve as editors on journals, and have been involved in faculty hiring. In addition, the PI will attend career development seminars offered by the Career and Postdoctoral Services office and Network for Women in Science at Scripps Florida, and will enroll in the Responsible Conduct of Research course. Therefore, with the hands-on, didactic and mentored training components, this plan will substantially enhance the PI's research skills in a new technique (optogenetics), permit her to use this technique under novel, innovative conditions (in conjunction with mouse self-administration), and advance her professional knowledge to ensure success and scientific independence to attain R01 funding in the early independent years as tenure-track faculty. PUBLIC HEALTH RELEVANCE: Tobacco addiction has a significant negative impact on the health and economic status of the individual and society. The proposed research will investigate the role of discrete brain pathways and neurotransmitter systems in mediating an inhibitory signal to nicotine that limits intake of the drug. Together, these data may reveal fundamental insights into the neurobiological mechanisms underlying nicotine reinforcement and dependence, and importantly, may lead to the development of novel therapeutics efficacious for smoking cessation in humans.