Dissecting neural circuits for breathing patterns

建议书摘要 呼吸是一种重要的功能，不断地受到来自身体的相互感觉信号的调节，众所周知，呼吸模式会影响情绪和认知过程。具有基本肺内感觉功能的呼吸模式，如叹息和咳嗽，与许多病理情况有关，包括睡眠呼吸暂停、婴儿猝死综合征、过度咳嗽、新冠肺炎和各种神经系统疾病，如恐慌症、恐惧症、创伤后应激障碍、药物滥用，甚至脑死亡。因此，迫切需要确定呼吸感觉间控制的神经机制，以及它们在病理条件下如何失效，以开发更有效的呼吸异常治疗方法。叹息是一种深呼吸，是在低氧中戏剧性地产生的两倍大小的吸气。相比之下，咳嗽是一种保护性的呼吸模式，其特征是呼气相扩大，由暴露在呼吸道中的咳嗽药剂触发。然而，这些基本的和离散的呼吸模式背后的神经回路，以及大脑如何解释和整合这些不同的感觉间感觉刺激，在很大程度上是未知的。在我们的初步研究中，我们在孤束核(NTS)中发现了两个具有不同基因表达、连接、神经活动和功能的神经元群体，NTS是大脑中第一个接受来自内脏器官的感觉间传入信号的中继中心。这些神经元分别介导低氧引起的叹息和咳嗽引起的咳嗽，这两种不同的呼吸模式与不同的内感信号有关。基于这些发现，我们建议检验我们的假设，即这两个不同的NTS神经元是两个分离的感觉间神经回路的关键节点，通过接收不同的传入输入和以不同的方式激活下游大脑回路，控制离散的呼吸模式并代表这些内部状态。我们将整合最先进的技术，包括基因靶向、基于病毒的神经回路追踪、活动依赖神经元靶向、光遗传学和化学遗传学、遗传消融、呼吸生理学、单分子荧光RNA原位杂交和体内钙记录，以确定在自由活动的小鼠体内这两个相互感受过程背后的神经回路和通路。通过聚焦于这两个不同的NTS神经元群体和神经回路，我们将描绘从外周到大脑的不同的感觉间传入路径，识别调节叹息和咳嗽的大脑区域，并定义解释和整合这些不同的感觉间信号的高级大脑区域。这项工作将为分别用于叹息和咳嗽的内感神经回路提供新的分子和细胞特异性，并揭示NTS和脑的组织原理，以确定迷走神经传入通路中不同的内感信号和输入。此外，拟议的研究还将为未来关于内感觉在神经病理条件下的作用的临床研究奠定基础。

Proposal Summary Breathing is a vital function constantly regulated by the interoceptive signals from the body, and breathing patterns are known to impact emotional and cognitive processes. Breathing patterns with essential pulmonary interoception functions, such as sighing and coughing, are relevant to many pathological conditions, including sleep apnea, sudden infant death syndrome, excessive coughing, COVID-19, and various nervous system disorders, such as panic disorder, phobias, post-traumatic stress disorder, drug abuse, and even brain death. Therefore, there is a critical need to identify the neural mechanisms underlying the interoceptive control of breathing and how they fail under pathological conditions, to develop more effective treatments to breathing abnormalities. Sighing is an augmented breath with a deep, double-size inspiration that is dramatically induced in hypoxia. In contrast, coughing is a protective breathing pattern with a characteristic enlarged expiration phase triggered by tussive agents exposed in the airways. However, the neural circuits underlying these essential and discrete breathing patterns and how the brain interprets and integrates these different interoceptive sensory stimuli are largely unknown. In our preliminary studies, we identified two neuronal populations with distinct gene expression, connectivity, neural activity, and function, in the nucleus of the solitary tract (NTS), the first relay center in the brain that receives interoceptive afferent signals from the visceral organs. These neurons respectively mediate hypoxia induced sighing and tussive challenge induced coughing, two discrete breathing patterns associated with different interoceptive signals. Based on these findings, we propose to test our hypothesis that these two distinct NTS neurons are the key nodes in two segregated interoceptive neural circuits for controlling discrete breathing patterns and for representing these internal states, by receiving distinct afferent inputs and differently activating downstream brain circuits. We will integrate state-of-the-art techniques, including genetic targeting, viral-based neural circuit tracing, activity dependent neuron targeting, optogenetics and chemogenetics, genetic ablation, respiratory physiology, single molecule fluorescent RNA in situ hybridization, and in vivo calcium recording, to identify the neural circuits and pathways underlying these two interoceptive processes in vivo in freely moving mice. By focusing on these two distinct NTS neuron populations and neural circuits, we will delineate the distinct interoceptive afferent pathways from the periphery to the brain, identify the brain regions that mediate sighing and coughing, and define the higher brain regions for interpreting and integrating these distinct interoceptive signals. This work will provide novel molecular and cellular specificity for the interoceptive neural circuits for sighing and coughing respectively, and reveal the organizing principles of the NTS and the brain to ascertain differential interoceptive signals and inputs through the vagal afferent pathways. Furthermore, the proposed studies will also build the foundation for future clinical studies on the role of interoception in neuropathological conditions.