A vagal-brainstem interoceptive circuit for cough-like defensive behaviors in mice

Respiratory defensive behaviors, like coughing, play a crucial role in protecting the respiratory system, ensuring its integrity and optimal function. How these critical behaviors are regulated by sensory stimuli within the body remains largely unknown. Here, we show that the nucleus of the solitary tract (NTS) in mice, a key hub in the brain for processing internal sensory signals and mediating interoceptive processes, contains heterogenous neuronal populations that differentially control breathing. Within these subtypes, activation of tachykinin 1 (Tac1) neurons triggers a specific respiratory behavior. Our detailed characterization of respiratory defensive behaviors reveals that these responses are cough-like behaviors. Chemogenetic silencing or genetic ablation of Tac1 neurons significantly reduces cough-like behaviors induced by tussive challenges. These Tac1 neurons receive synaptic inputs from the bronchopulmonary chemosensory and mechanosensory neurons in the vagal ganglion, and directly integrate the medullary regions to control sequential phases of cough-like defensive behaviors. We propose that these Tac1 neurons are a key component of the airway-vagal-brain neural circuit that controls cough-like defensive behaviors in mice, and they coordinate the downstream modular circuits to elicit the sequential motor pattern of forceful expiratory responses. Five mice at postnatal day 5 were euthanized by isoflurane inhalation followed by decapitation. The brain was isolated and embedded in 4% agarose II in artificial cerebrospinal fluid (ACSF) and sectioned at 200um thickness on a VT 1000S vibratome (Leica). The caudal NTS tissue and adjacent regions in the dorsal vagal complex were manually dissected based on the morphology (the relative position to area postrema, 4th ventricle, and central canal), and referred as the NTS. Tissue was pulled together in hibernate A solution (Gibco), followed by digestion with 20 u/ml papain (Worthington Biochemical Corporation) and serial trituration by Pasteur pipets to generate single cell suspension. The cells were stained with 0.2% DyeCycle Ruby (Invitrogen) at 37 degree for 15 minutes before passing through a 70um cell strainer (Falcon). The live cell suspensions were sorted and collected by a BD FACSAria III Cell Sorter (BD Biosciences) with a nozzle diameter of 100 um. The FACS sample was loaded to the 10X Chromium platform for capturing. The following cDNA synthesis with PCR and library preparation were done according to the manufacturer’s protocol. Sequencing was performed on a NovaSeq sequencer (Illumina)

咳嗽等呼吸道防御行为对保护呼吸系统、维持其结构完整与最佳功能至关重要。目前学界对于这类关键行为如何受体内感觉刺激调控的认知仍十分有限。本研究证实，小鼠孤束核（nucleus of the solitary tract, NTS）——这一大脑内处理内部感觉信号、介导内感受过程的关键枢纽——包含异质性神经元群，其可差异化调控呼吸活动。在这些神经元亚型中，速激肽1（tachykinin 1, Tac1）神经元的激活会触发特定的呼吸行为。

我们对呼吸道防御行为的详细表征显示，这类行为属于类咳嗽行为。通过化学遗传学沉默或基因敲除Tac1神经元，可显著降低由致咳刺激诱导的类咳嗽行为。这类Tac1神经元接收来自迷走神经节内支气管肺化学感受性与机械感受性神经元的突触输入，并直接整合延髓区域活动，以调控类咳嗽防御行为的时序阶段。

我们提出，Tac1神经元是小鼠体内调控类咳嗽防御行为的气道-迷走神经-大脑神经环路的关键组成部分，它们可协调下游模块化环路，以引发有力呼气反应的时序运动模式。

本研究取5只出生后第5天的小鼠，经异氟烷吸入麻醉后处死并断头。分离脑组织后，将其包埋于含4%琼脂糖II的人工脑脊液（artificial cerebrospinal fluid, ACSF）中，使用VT 1000S振动切片机（Leica）切成200 μm厚的脑片。根据形态学特征（相对于最后区、第四脑室与中央管的相对位置），手动分离背侧迷走复合体中的尾侧孤束核组织及相邻区域，即本研究定义的孤束核。将组织收集于Hibernate A培养基（Gibco）中，随后用20 u/ml的木瓜蛋白酶（Worthington Biochemical Corporation）消化，并通过巴斯德吸管反复吹打制备单细胞悬液。将细胞置于37℃下用0.2% DyeCycle Ruby染料（Invitrogen）染色15分钟，随后通过70 μm细胞筛（Falcon）过滤。使用喷嘴直径100 μm的BD FACSAria III型细胞分选仪（BD Biosciences）分选并收集活细胞悬液。将分选得到的样本加载至10X Chromium平台进行细胞捕获。后续的cDNA合成、PCR扩增与文库制备均严格按照制造商的操作流程进行。测序工作在NovaSeq测序仪（Illumina）上完成。