Ionic and Structural Mechanisms for Sensory Neuromodulation of the Esophagus

项目摘要 食管感觉（传入）神经对于调节食管感觉-运动功能是必需的， 导致许多临床症状（吞咽困难、吞咽痛、恶心、非心源性胸痛、难治性 胃灼热等）在疾病中受到刺激或失调。因此，食管的神经调节 预期传入神经在许多情况下提供缓解。神经调节的研究进展 由于我们对食管传入神经的关键方面的了解有限， 神经生物学在这个为期三年的提案中，我们将重点关注两个高度相关的未充分研究的 地区第一，神经末梢在不同食管隔室中的位置。这对于以下方面至关重要： 了解它们在食管疾病中的作用，以及神经调节的设计和放置 接口。第二，我们将获得数据，这将促进我们对离子通道潜在作用的理解 食管传入神经的潜在传导，这将为电生理的发展和优化提供信息。 神经调节策略我们将不仅在豚鼠和小鼠中解决这些问题， 食管和从人供体获得的分离的食管神经。目标1将阐明 食管中C纤维亚型的神经末梢和轴突。我们假设神经嵴 源自基板的C-纤维支配不同的食管组织隔室（分别为粘膜与肌肉）。 我们将通过选择性地观察基板与神经嵴来源的轴突来解决我们的假设， 用选择性AAV-病毒载体-GPF转染豚鼠和转基因小鼠， 在人供体全食管中的发现。目的2：获得完整的食管反流镜 传入神经类型集中于介导动作电位传导的离子通道（尤其是NaV和 KV）。我们假设神经嵴源性（迷走神经颈静脉和脊髓背根神经节）C纤维的转录本 神经元与基板衍生的结状C纤维和机械感受器（包括 NaV和KV）。我们将对从食管逆行标记的神经元进行深度RNAseq分析， TRPV 1-tdTomato小鼠中表达，并通过qRT-PCR验证Nav和Kv的表达。我们还将分析迷走神经 食蟹猴的结状核和颈静脉神经元。目的3将评估离子通道的作用， 食管传入神经类型的动作电位传导。我们将首先评估 各种NaV（NaV 1.1-NaV 1.9）亚型的作用，以及后来对KV通道的作用。我们假设传导 在食管传入神经类型中，不仅由Nav1.7介导，而且还由其他河豚毒素（TTX）敏感的 通道，可能在神经嵴和基板衍生的C纤维之间存在差异（NaV1.1/1.6 vs. NaV1.2/1.3）。 这将在豚鼠和小鼠神经支配食管制备中使用药理学工具进行研究 和shRNA敲低，以及人类食管神经。

Project Summary Esophageal sensory (afferent) nerves are essential for regulation of esophageal sensory-motor function and contribute to many clinical symptoms (dysphagia, odynophagia, nausea, non-cardiac chest pain, refractory heartburn, etc.) when stimulated or dysregulated in diseases. Therefore, the neuromodulation of esophageal afferent nerves is expected to provide relief in many conditions. Progress in advancing neuromodulatory approaches targeting esophageal afferent nerves is hampered by our limited understanding of key aspects of their neurobiology. In this three-year proposal we will focus on two such highly relevant understudied areas. First, the location of the nerve terminals within different esophageal compartments. This is essential for understanding of their role in esophageal diseases, and for design and placement of neuromodulation interfaces. Second, we will obtain data that will advance our understanding of the ion channels underlying action potential conduction in esophageal afferent nerves, which will inform development and optimization of electrical neuromodulation strategies. We will address these issues not only in the guinea pig and mouse but also in the esophagus and isolated esophageal nerves obtained from human donors. Aim 1 will elucidate the location of nerve terminals and axons of C-fiber subtypes in the esophagus. We hypothesize that the neural crest- and placodes-derived C-fibers innervate distinct esophageal tissue compartments (mucosa vs. muscle, respectively). We will address our hypothesis by selective visualization of placodes- vs. neural crest-derived axon and terminals with selective AAV-virus vector-GPF transfection in guinea pig and transgenic mice and evaluate key findings in human donor whole esophagi. In Aim 2, we will obtain the complete trascriptomes of esophageal afferent nerve types focusing on ion channels that mediate action potential conduction (especially NaVs and KVs). We hypothesize that the trascriptomes of the neural crest-derived (vagal jugular and spinal DRG) C-fiber neurons are similar but distinct from those of placodes-derived nodose C-fibers and mechanosensors (including NaVs and KVs). We will perform deep RNAseq analysis on neurons retrogradely labeled from the esophagus in TRPV1-tdTomato mice and validate the expression of Nav and Kv by qRT-PCR. We will also analyze vagal nodose and jugular neurons in Cynomolgus monkey. Aim 3 will evaluate the role of ion channels underlying action potential conduction in esophageal afferent nerve types by electrophysiology. We will initially evaluate the role of the various NaV (NaV1.1 –NaV 1.9) subtypes, and later on KV channels. We hypothesize that conduction in esophageal afferent nerve types is mediated not only by Nav1.7, but also other tetrodotoxin (TTX)-sensitive channels, possibly distinct between the neural crest- and placodes-derived C-fibers (NaV1.1/1.6 vs. NaV1.2/1.3). This will be investigates in guinea pig and mouse innervated esophagus preparation using pharmacological tools and shRNA knockdown, and in human esophageal nerves.