Ion Channel Mechanisms of Inspiratory Breathing Movements in Mice

小鼠吸气呼吸运动的离子通道机制

• 批准号: 10357582
• 负责人: Christopher A. Del Negro
• 金额: $ 27.84万
• 依托单位: COLLEGE OF WILLIAM AND MARY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357582
• 关键词: Acute; Adult; Alpha Rhythm; Attenuated; Behavior; Biophysics; Brain; Brain Stem; Breathing; Cations; Cells; Charge; Chest; Complex; Cre driver; Etiology; Gene Expression; Generations; Genes; Genetic; Goals; Health; Human; Hypoxia; In Vitro; Interneurons; Ion Channel; Knock-out; Knockout Mice; Knowledge; LoxP-flanked allele; Lung; Mammals; Measurement; Mediating; Motor; Movement; Mus; Muscle; Neonatal; Neurobiology; Neurodegenerative Disorders; Neurons; Neurosciences; Occupations; Pacemakers; Pathology; Pattern; Periodicity; Pharmacology; Phenotype; Physiology; Population; Potassium Channel; Property; Prophylactic treatment; Respiration; Respiration Disorders; Role; SCN8A gene; Sampling; Site; Slice; Synapses; TRP channel; Techniques; Technology; Testing; awake; comparative; genetic technology; in vivo; insight; knock-down; mature animal; models and simulation; mouse genetics; neurophysiology; next generation; patch clamp; preBotzinger complex; premature; progenitor; receptor; relating to nervous system; respiratory; sensory feedback; small hairpin RNA; spatiotemporal; success; transcriptome sequencing

PROJECT SUMMARY Our goal is to explain the neurophysiology of breathing. Breathing refers to periodic movements of the chest and airways that ventilate the lungs. To breathe, the brain must:  Generate a rhythm,  Produce a spatiotemporal pattern for breathing muscles,  Integrate sensory feedback. Here we focus on jobs 1 and 2, discovering the ionic mechanisms underlying the rhythm and motor pattern. The brainstem preBötzinger complex (preBötC) generates the inspiratory breathing rhythm. Its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors (i.e., Dbx1 neurons). Because we know the site (preBötC) and the canonical cell-class (Dbx1) at the point of origin for breathing, we are well equipped to discover which ion channels influence normal inspiration (eupnea), ‘sighs’, and gasps in hypoxia. We manipulate ion channels using genetic technologies. We assess breathing phenotypes in intact adult mice. We explain the biophysics of these phenotypes via patch-clamp recordings from adult Dbx1 preBötC neurons. Three types of ion channels are implicated in preBötC function: (1) Na+ channels that engender persistent Na+ current (INaP), (2) Transient receptor potential (Trp) channels that mediate Ca2+-activated nonspecific cationic current (ICAN), and (3) K+ channels that give rise to transient outward K+ current (i.e., A-current, IA). Specific aim 1 evaluates the role of INaP in Dbx1 preBötC neurons. A rhythmogenic role for INaP has been suspected for 27 years. We use intersectional mouse genetics and short-hairpin RNA (shRNA) to knockout or knock-down Na+ channel genes that give rise to INaP and evaluate its role in eupnea, sighing, and gasping. Specific aim 2 evaluates the role of ICAN in Dbx1 preBötC neurons. ICAN is a major charge carrier for inspiratory bursts. Next-generation RNA Seq technology provides us with a suite of Trp channel targets that we will acutely attenuate using shRNA to test the role of ICAN in eupnea, sighing, and gasping. Specific aim 3 evaluates the role of IA in Dbx1 preBötC neurons. IA-expressing preBötC neurons feature other key rhythmogenic properties so they may be specialized. We use intersectional mouse genetics and shRNA to knockout or knock-down K+ channel genes for IA to evaluate its role in eupnea, sighing, and gasping. Then we selectively kill the 56% of IA-expressing Dbx1 preBötC neurons to test whether they serve a specialized rhythmogenic role. The success of this project would be a watershed in terms of understanding the ion channel-level origins of real behavior. The new knowledge could be applicable to treatment and prophylaxis of respiratory pathologies with a central etiology. The new knowledge may provide general insights regarding the neural origins of motor rhythms.

项目摘要 我们的目标是解释呼吸的神经生理学。呼吸是指胸部的周期性运动 和覆盖肺部的呼吸道为了呼吸，大脑必须： 产生一种节奏， 为呼吸肌肉产生时空模式， 整合感官反馈。 在这里，我们专注于工作1和2，发现节奏和运动模式背后的离子机制。 脑干前波青格复合体（preBötC）产生吸气呼吸节律。其核心 节律性中间神经元来源于Dbx 1表达祖细胞（即，Dbx1神经元）。因为我们 知道呼吸起源点的位置（preBötC）和规范细胞类（Dbx1），我们很好地 配备发现哪些离子通道影响正常的吸气（正常呼吸），“叹息”，并在缺氧喘息。 我们用基因技术操纵离子通道。我们评估完整的成年小鼠的呼吸表型。 我们解释这些表型的生物物理通过膜片钳记录从成人Dbx1 preBötC神经元。 三种类型的离子通道参与preBötC功能：（1）产生持续Na+的Na+通道 电流（INaP），（2）瞬时受体电位（Trp）通道，介导Ca 2+激活的非特异性阳离子 电流（ICAN），和（3）引起瞬时外向K+电流的K+通道（即，A电流，IA）。 具体目标1评估INaP在Dbx 1 preBötC神经元中的作用。INaP的节律性作用已经被证实， 怀疑了27年。我们使用交叉小鼠遗传学和短发夹RNA（shRNA）敲除或 敲低Na+通道基因，引起INaP，并评估其在正常呼吸、叹息和喘息中的作用。 具体目标2评估ICAN在Dbx1 preBötC神经元中的作用。ICAN是吸气的主要电荷载体。 突发下一代RNA Seq技术为我们提供了一套Trp通道靶点， 使用shRNA急性减弱以测试ICAN在正常呼吸、叹息和喘息中的作用。 具体目标3评估了IA在Dbx1 preBötC神经元中的作用。表达IA的preBötC神经元具有其他 关键的节律性特征，因此它们可能是特化的。我们使用交叉小鼠遗传学和shRNA， 敲除或敲低IA的K+通道基因，以评估其在正常呼吸、叹气和喘息中的作用。然后我们 选择性地杀死56%的IA表达Dbx1 preBötC神经元，以测试它们是否为一个专门的 节奏发生作用 这个项目的成功将是理解离子通道水平起源的分水岭。 真实的行为。新的知识可能适用于治疗和预防呼吸道疾病 有一个中心病因。新的知识可能会提供关于运动神经起源的一般见解 节奏

项目成果

KCNQ Current Contributes to Inspiratory Burst Termination in the Pre-Bötzinger Complex of Neonatal Rats in vitro.

• DOI: 10.3389/fphys.2021.626470
• 发表时间: 2021
• 期刊: Frontiers in physiology
• 影响因子: 4
• 作者: Revill AL;Katzell A;Del Negro CA;Milsom WK;Funk GD
• 通讯作者: Funk GD

Transcriptomes of electrophysiologically recorded Dbx1-derived respiratory neurons of the preBötzinger complex in neonatal mice.

• DOI: 10.1038/s41598-022-06834-z
• 发表时间: 2022-02-21
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Kallurkar PS;Picardo MCD;Sugimura YK;Saha MS;Conradi Smith GD;Del Negro CA
• 通讯作者: Del Negro CA

Single cell transcriptome sequencing of inspiratory neurons of the preBötzinger complex in neonatal mice.

• DOI: 10.1038/s41597-022-01569-y
• 发表时间: 2022-07-30
• 期刊: SCIENTIFIC DATA
• 影响因子: 9.8
• 作者: David, Caroline K.;Sugimura, Yae K.;Kallurkar, Prajkta S.;Picardo, Maria Cristina D.;Saha, Margaret S.;Conradi Smith, Gregory D.;Del Negro, Christopher A.
• 通讯作者: Del Negro, Christopher A.