Nicotine & Nodose: A Neural Basis for Peripheral Control over Nicotine Intake

摘要 在美国，以习惯性烟草使用为形式的尼古丁成瘾是导致过早死亡的主要原因。 美国每年至少花费1700亿美元的医疗保健相关费用。除了令人上瘾之外 尼古丁会引发过多的呼吸道、心血管、胃肠道和免疫系统疾病， 不仅在大脑中，而且在吸烟者的身体中也反映出它的作用。虽然公认的是， 尼古丁的成瘾特性与其对烟碱乙酰胆碱的直接药理作用有关 位于奖励和动机脑回路的受体，我们实验室的证据表明， 尼古丁与其对后脑厌恶回路（例如孤束核（NTS）， 脚间核和内侧缰核）在调节尼古丁摄入方面也起着重要作用。 然而，目前尚不清楚尼古丁是否仅通过直接作用于中枢表达的尼古丁受体而作用于这些回路。 受体，或者如果尼古丁也间接通过迷走神经（结状神经节，NG）衍生的感觉输入起作用， 主要是在NTS。在这个拨款申请中提出的初步数据强烈表明，外周神经系统的作用， 尼古丁在控制尼古丁摄入方面的作用。例如，外周限制的完全尼古丁激动剂， 甲基烟碱引起的条件性位置厌恶超过等摩尔剂量的烟碱所产生的厌恶。 此外，外周限制性胆囊收缩素受体（CCKR）激动剂CCK-8（10 µ*kg-1）降低了 自愿尼古丁摄入量，特别是在焦虑尼古丁剂量。根据这些初步数据和 观察到啮齿类动物和人类的血浆CCK水平因尼古丁而失调，我推测， 肠神经支配NG神经元中的CCKRs增强从外周到NTS的厌恶性尼古丁信号，从而 调节尼古丁摄入量我将使用尼古丁静脉自我给药小鼠模型来检验这一假设 与NG、FosTRAP小鼠和化学遗传学的CCKR特异性病变组合。然后我将定义 使用单细胞RNA测序来确定NG对厌恶剂量的尼古丁的转录响应性。 最后，我将采用体内CRISPR-Cas9介导的基因组切割策略， 尼古丁反应基因在NG和评估尼古丁摄入的后果。完成这一高度 创新的建议将大大提高我在老鼠遗传学、外科手术和计算机方面的技能 编程，并为我提供了全新的单细胞测序，生物信息学和先进的培训， 基因组编辑技术。它也将有助于目前关于感官如何 与尼古丁作用有关的外周信息通过迷走神经传递到后脑。限定 这样的机制将使我在令人兴奋和迅速发展的领域中获得成功的独立职业生涯 大脑与身体的相互作用总之，本培训计划将为我提供概念、技术和 这些学习经历将成为我过渡到独立研究调查员的跳板。

ABSTRACT Nicotine addiction in the form of habitual tobacco use is the leading cause of premature death in the United States and costs at least $170 billion in healthcare-related expenses each year. In addition to its addictive qualities, nicotine triggers a plethora of respiratory, cardiovascular, gastrointestinal, and immune disorders, reflecting its actions in not only the brain, but also in the body of smokers. While it is well established that the addictive properties of nicotine are related to its direct pharmacological actions on nicotinic acetylcholine receptors located in reward and motivation brain circuits, evidence from our lab suggests that noxious effects of nicotine related to its actions on hindbrain aversion circuits (e.g. nucleus of the solitary tract (NTS), interpeduncular nucleus, and medial habenula) play a significant role in regulating nicotine intake as well. However, it is unclear if nicotine acts solely on these circuits by direct action on centrally expressed nicotinic receptors or if nicotine also acts indirectly via vagally (nodose ganglia, NG) derived sensory inputs that terminate primarily at the NTS. Preliminary data presented in this grant application strongly suggest a role for peripheral actions of nicotine in controlling nicotine intake. For instance, the peripherally-restricted, full nicotine agonist, methylnicotinium causes a conditioned place aversion beyond that generated by an equimolar dose of nicotine. Further, the peripherally-restricted cholecystokinin receptor (CCKR) agonist, CCK-8 (10 µ*kg-1) decreased volitional nicotine intake, especially at anxiogenic nicotine doses. Given these preliminary data and the observation that plasma CCK levels are dysregulated by nicotine in rodents and humans, I hypothesize that CCKRs in gut-innervating NG neurons potentiate aversive nicotine signals from the periphery to the NTS thereby regulating nicotine intake. I will test this hypothesis using a nicotine intravenous self-administration mouse model in combination with CCKR-specific lesions of the NG, FosTRAP mice, and chemogenetics. I will then define the transcriptional responsiveness of the NG to an aversive dose of nicotine using single cell RNA sequencing. Finally, I will employ an in vivo CRISPR-Cas9-mediated genomic cleavage strategy to knockdown prioritized nicotine-response genes in the NG and assess the consequences on nicotine intake. Completion of this highly innovative proposal will substantially advance my technical skills in mouse genetics, surgery, and computer programing, and provide me with entirely new training in single cell sequencing, bioinformatics, and advanced genome editing technologies. It will also contribute to the currently sparse literature about how sensory information related to nicotine actions in the periphery are transmitted via the vagus to the hindbrain. Defining such a mechanism will position me for a successful independent career in the exciting and rapidly growing field of brain-body interactions. In summation, this training plan will provide me with the conceptual, technical, and learning experiences that will serve as a springboard for my transition to an independent research investigator.