Role of TRP Channels in Respiratory Rhythm and Breathing

 描述（由申请人提供）：我们的目的是解释呼吸行为的神经起源。呼吸控制回路的功能障碍会导致严重的健康问题，包括阻塞性和中枢性呼吸暂停，以及呼吸衰竭和死亡。这些病症折磨早产儿、儿童、成人和患有神经退行性疾病的患者。该项目的重点是脑干前BötC复合体（preBötC）中吸气呼吸运动的核心节律产生回路。preBötC已经被研究了25年。它的定义特征及其组成节律产生神经元已被详细记录，然而，在preBötC神经元中产生吸气爆发的离子通道的分子身份仍然没有确定。本提案的具体目标是解决这一关键的知识差距。如果我们成功了，那么呼吸将是第一个可以在网络、细胞和离子通道水平上解释的哺乳动物运动行为。特别是，这个项目将测试一个主要的假设，一类非选择性阳离子通道产生吸气爆发，并通过扩展，这些通道的基础上的电机驱动呼吸在活的动物。人们普遍认为，一个分子身份不明的Ca2+激活的非选择性阳离子电流（ICAN）有助于前BötC神经元吸气爆发的产生。基于来自多个来源的证据，瞬时受体电位（TRP）超家族的非选择性阳离子通道是在preBötC中产生ICAN的通道的最佳候选者。我们有三个主要目标。1)使用定量PCR、生理学和免疫组织化学确定哪些TRP通道在preBötC神经元中表达。2)下调这些TRP通道干扰mRNA转录和过表达显性负通道突变体的节奏活跃的切片文化。然后，我们将测量preBötC神经元的爆发产生能力受到损害的程度。3)下调活体小鼠preBötC中相同的TRP通道，并测量呼吸运动受损的程度。该项目通过使用当代分子生物学技术来解决迄今为止主要由电生理学解决的问题。该项目意义重大，因为它确定了负责吸气爆发产生、聚集运动驱动和真实的呼吸行为的离子通道，这代表了我们理解的变革性进步，将为对抗呼吸道疾病的新预防和治疗策略提供信息。PI是这项工作的理想科学家，因为他是呼吸神经生物学的领导者，他首先描述了ICAN以及核心节律前BötC神经元的遗传特性。如果这个项目成功，那么我们将了解吸气爆发产生的分子水平机制。然后，医学科学将能够在治疗上靶向这些通道，以改善许多情况下的呼吸道病变。神经科学将最终知道所有呼吸生理学的基本分子起源点。

 DESCRIPTION (provided by applicant): We aim to explain the neural origins of breathing behavior. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project focuses on the core rhythm-generating circuit for inspiratory breathing movements in the pre-Bötzinger complex (preBötC) of the brainstem. The preBötC has been studied for 25 years. Its defining characteristics and its constituent rhythm-generating neurons have been documented in some detail, however, the molecular identity of the ion channels in preBötC neurons that generate inspiratory bursts remains unidentified. The specific aims of this proposal address this key knowledge gap. If we succeed then breathing would be the first mammalian motor behavior that can be explained at the level of networks, cells, and ion channels. In particular, this projec will test one leading hypothesis that a class of non-selective cation channels generates inspiratory bursts, and by extension, that these channels underlie the motor drive for breathing in living animals. It is widely acknowledged that a molecularly unidentified Ca2+-activated non-selective cationic current (ICAN) contributes to inspiratory burst generation in preBötC neurons. Non-selective cation channels of the transient receptor potential (TRP) superfamily are the best candidates for the channel(s) that give rise to ICAN in the preBötC based on evidence from several sources. We have three main objectives. 1) Determine which TRP channels are expressed in preBötC neurons using quantitative PCR, physiology, and immunohistochemistry. 2) Down-regulate these TRP channels by interfering with mRNA transcripts and overexpressing dominant negative channel mutants in rhythmically active slice cultures. Then, we will measure the degree to which the burst-generating capability of preBötC neurons is compromised. 3) Down-regulate the same TRP channels in the preBötC of living mice and measure the extent to which breathing movements are impaired. This project innovates by using contemporary molecular biology techniques to address a problem that has heretofore been approached primarily by electrophysiology. This project is significant because it identifies the ion channels responsible for inspiratory burst generation, aggregate motor drive, and real breathing behavior, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who firt characterized ICAN as well as the genetic identity of the core rhythmogenic preBötC neurons. If this project succeeds, then we will know the molecular-level mechanism for inspiratory burst generation. Medical science would then be able to therapeutically target those channels to ameliorate respiratory pathologies in many circumstances. Neuroscience would finally know the fundamental molecular point of origin for all respiratory physiology.