Ionic and Structural Mechanisms for Sensory Neuromodulation of the Esophagus

食管感觉神经调节的离子和结构机制

• 批准号: 9769712
• 负责人: Thomas Edward Taylor-Clark
• 金额: $ 24.55万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769712
• 关键词: Acids; Action Potentials; Address; Agonist; Anatomy; Area; Axon; C Fiber; Cavia; Chemicals; Chest Pain; Clinical; Communication; Data; Deglutition; Deglutition Disorders; Development; Disease; Electrophysiology (science); Embryo; Esophageal; Esophageal Diseases; Esophageal Tissue; Esophagus; Esophagus motility; Esthesia; Frequencies; Heartburn; Histology; Human; Imagery; Inflammation Mediators; Investigation; Ion Channel; Knowledge; Label; Lead; Location; Lung diseases; Macaca fascicularis; Mediating; Motor; Mucous Membrane; Mus; Muscle; Myenteric Plexus; Nausea; Nerve; Neural Crest; Neurobiology; Neurons; Nociception; Patients; Pattern; Pharmacological Treatment; Pharmacology; Play; Preparation; Presynaptic Terminals; Property; Publishing; Quantitative Reverse Transcriptase PCR; Reflex action; Reflex control; Refractory; Regulation; Role; SCN1A protein; Sensory; Sodium Channel; Sphincter; Spinal; Stimulus; Structure; Symptoms; TRPV1 gene; Tetrodotoxin; Transfection; Transgenic Mice; Virus; afferent nerve; base; design; gastrointestinal system; insight; knock-down; neurophysiology; neuroregulation; novel; pain sensation; painful swallowing; receptor; sensory mechanism; small hairpin RNA; tool; transcriptome sequencing; vector; voltage gated channel

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.

项目概要 食管感觉（传入）神经对于调节食管感觉运动功能和 导致许多临床症状（吞咽困难、吞咽痛、恶心、非心源性胸痛、难治性胸痛） 胃灼热等）在疾病中受到刺激或失调时。因此，食管神经调节 传入神经有望在许多情况下提供缓解。神经调节研究进展 针对食管传入神经的方法因我们对食管传入神经关键方面的了解有限而受到阻碍 他们的神经生物学。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待研究项目 地区。首先，不同食管腔内神经末梢的位置。这对于 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。其次，我们将获得有助于加深我们对离子通道潜在作用的理解的数据 食管传入神经的潜在传导，这将有助于电的发展和优化 神经调节策略。我们不仅会在豚鼠和小鼠身上解决这些问题，还会在 从人类捐赠者处获得食管和分离的食管神经。目标 1 将阐明 食管中 C 纤维亚型的神经末梢和轴突。我们假设神经嵴和 基板衍生的 C 纤维支配不同的食管组织区室（分别为粘膜与肌肉）。 我们将通过选择性可视化基板与神经嵴衍生的轴突来解决我们的假设 选择性 AAV 病毒载体-GPF 转染豚鼠和转基因小鼠的终端并评估关键 在人类捐赠者整个食管中的发现。在目标 2 中，我们将获得食管的完整转录组 传入神经类型专注于介导动作电位传导的离子通道（尤其是 NaV 和 KV）。我们假设神经嵴衍生的（迷走颈静脉和脊髓 DRG）C 纤维的转录组 神经元与基板衍生的结状 C 纤维和机械传感器（包括 NaV 和 KV）。我们将对食管逆行标记的神经元进行深度 RNAseq 分析 TRPV1-tdTomato 小鼠并通过 qRT-PCR 验证 Nav 和 Kv 的表达。我们还将分析迷走神经 食蟹猴的结状神经元和颈静脉神经元。目标 3 将评估离子通道的作用 通过电生理学研究食管传入神经类型的动作电位传导。我们将初步评估 各种 NaV (NaV1.1 –NaV 1.9) 亚型的作用，以及后来的 KV 通道。我们假设传导 在食管传入神经类型中，不仅由 Nav1.7 介导，还由其他河豚毒素 (TTX) 敏感的介导 通道，可能在神经嵴和基板衍生的 C 纤维之间有所不同（NaV1.1/1.6 与 NaV1.2/1.3）。 这将使用药理学工具在豚鼠和小鼠神经支配的食管制备中进行研究 和 shRNA 敲低，以及人类食管神经。

项目成果

Stimulus intensity-dependent recruitment of NaV1 subunits in action potential initiation in nerve terminals of vagal C-fibers innervating the esophagus.

NaV1 亚基在支配食管的迷走神经 C 纤维神经末梢的动作电位启动中的刺激强度依赖性募集。

• DOI: 10.1152/ajpgi.00122.2019
• 发表时间: 2020
• 期刊: American journal of physiology. Gastrointestinal and liver physiology
• 作者: Ru,Fei;Pavelkova,Nikoleta;Krajewski,JeffreyL;McDermott,JeffS;Undem,BradleyJ;Kollarik,Marian
• 通讯作者: Kollarik,Marian