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.

项目摘要 食管感觉（传入）神经对于调节食道运动运动功能和 有助于许多临床症状（吞咽困难，耳尾，恶心，非心脏胸痛，难治性 当疾病刺激或失调时，胃灼热等。因此，食管的神经调节 预计在许多情况下，传入的神经会缓解。推进神经调节的进展 靶向食道传入神经的方法受到我们对关键方面的有限理解的阻碍 他们的神经生物学。在这个为期三年的建议中，我们将重点关注两个这样的高度相关的理解 区域。首先，神经末端在不同食管室内的位置。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，我们将获取将提高我们对动作基本离子渠道的理解的数据 食管传入神经的潜在传导，这将为电气开发和优化提供信息 神经调节策略。我们将不仅在几内亚猪和鼠标中解决这些问题 从人类供体获得的食道和孤立的食管神经。 AIM 1将阐明 食管中C纤维亚型的神经末端和轴突。我们假设神经波峰和 放置的c纤维支配不同的食管组织室（分别为粘膜和肌肉）。 我们将通过选择性可视化位置来解决我们的假设： 豚鼠和转基因小鼠中具有选择性AAV-VIRUS VIRUS载体-GPF转染的末端以及评估钥匙 人类供体全食管中的发现。在AIM 2中，我们将获得食管的完整曲线。 关注介导动作潜在传导的离子渠道的传入神经类型（尤其是NAV和 KVS）。我们假设神经rest衍生的（迷走神经和脊柱DRG）C纤维的曲线图 神经元相似，但与放置的淋巴结c纤维和机械学的神经元不同（包括 NAVS和KVS）。我们将对从食道上逆行标记的神经元进行深入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 cynomolgus猴子中的nodose和颈神经元。 AIM 3将评估基础离子渠道的作用 通过电生理学对食管传入神经类型的动作潜在传导。我们最初将评估 各种NAV（NAV1.1 –NAV 1.9）子类型的角色，然后在KV通道上。我们假设该传导 在食道中，神经类型不仅是由NAV1.7介导的，而且还介导其他四毒素（TTX）敏感 通道，可能在神经rest和放置的C纤维之间有所不同（NAV1.1/1.6 vs. NAV1.2/1.3）。 这将在豚鼠和小鼠神经支配的食管制剂中进行研究 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