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

摘要 习惯性吸烟的尼古丁成瘾是美国人过早死亡的主要原因 美国各州每年至少花费1700亿美元与医疗保健相关的费用。除了它令人上瘾之外 尼古丁会引发过多的呼吸系统、心血管、胃肠道和免疫系统疾病， 不仅反映在大脑中，也反映在吸烟者的身体中。虽然公认的是， 尼古丁的成瘾特性与其对烟碱型乙酰胆碱的直接药理作用有关 位于奖赏和动机大脑回路的受体，来自我们实验室的证据表明， 尼古丁与其在后脑厌恶回路(如孤束核(NTS)， 脚间核和内侧缰核)在调节尼古丁摄取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来作用于这些环路。 受体或尼古丁是否也通过迷走神经(结状神经节，NG)间接作用于终止的感觉输入 主要是在新界南区。这项拨款申请中提供的初步数据有力地表明了外围设备的作用 尼古丁在控制尼古丁摄入量中的作用。例如，外周受限的完全尼古丁激动剂， 除了等摩尔剂量的尼古丁外，甲基尼古丁还会引起条件性位置厌恶。 此外，外周限制性的CCKR激动剂CCK-8(10µ*kg-1)减少 自愿摄入尼古丁，尤其是在引起焦虑的尼古丁剂量下。鉴于这些初步数据和 观察到啮齿动物和人类的血浆CCK水平受到尼古丁的失调调节，我假设 支配肠道的NG神经元中的CCKRs增强了从外周到NTS的厌恶尼古丁信号，从而 控制尼古丁摄入量。我将使用尼古丁静脉注射自我给药的小鼠模型来验证这一假设。 与CCKR特异的NG、FosTRAP小鼠和化学遗传学病变相结合。然后，我将定义 用单细胞RNA测序法研究NG对不同剂量尼古丁的转录反应。 最后，我将采用体内CRISPR-Cas9介导的基因组切割策略来优先击倒 并评估其对尼古丁摄入量的影响。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术技能 编程，并为我提供了在单细胞测序、生物信息学和高级 基因组编辑技术。这也将有助于目前稀少的关于感官如何 与外周尼古丁活动有关的信息通过迷走神经传递到后脑。定义 这样的机制将使我能够在令人兴奋和快速增长的领域取得成功的独立职业生涯 大脑与身体的相互作用。综上所述，本培训计划将为我提供概念、技术和 学习经验将成为我向独立研究调查员过渡的跳板。

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.