Molecularly defined circuits for cardiovascular and cardiopulmonary control

The sympathetic and parasympathetic nervous systems regulate the activities of internal organs, but the molecular and functional diversity of their constituent neurons and circuits remains largely unknown. Here we use retrograde neuronal tracing, single-cell RNA sequencing, optogenetics and physiological experiments to dissect the cardiac parasympathetic control circuit in mice. We show that cardiac-innervating neurons in the brainstem nucleus ambiguus (Amb) are comprised of two molecularly, anatomically and functionally distinct subtypes. The first, which we call ambiguus cardiovascular (ACV) neurons (approximately 35 neurons per Amb), define the classical cardiac parasympathetic circuit. They selectively innervate a subset of cardiac parasympathetic ganglion neurons and mediate the baroreceptor reflex, slowing heart rate and atrioventricular node conduction in response to increased blood pressure. The other, ambiguus cardiopulmonary (ACP) neurons (approximately 15 neurons per Amb) innervate cardiac ganglion neurons intermingled with and functionally indistinguishable from those innervated by ACV neurons. ACP neurons also innervate most or all lung parasympathetic ganglion neurons--clonal labelling shows that individual ACP neurons innervate both organs. ACP neurons mediate the dive reflex, the simultaneous bradycardia and bronchoconstriction that follows water immersion. Thus, parasympathetic control of the heart is organized into two parallel circuits, one that selectively controls cardiac function (ACV circuit) and another that coordinates cardiac and pulmonary function (ACP circuit). This new understanding of cardiac control has implications for treating cardiac and pulmonary diseases and for elucidating the control and coordination circuits of other organs. Single cell mRNA profiles were generated from mouse nucleus ambiguus neurons that were retrogradely labeled from the heart or larynx. Mice were postnatal day 2 to 4 at time of sequencing. cDNA was generated using the Smart-seq2 protocol.交感与副交感神经系统调控内脏器官的活动，但其组成神经元与神经环路的分子及功能多样性在很大程度上仍未被充分阐明。本研究通过逆行神经元追踪（retrograde neuronal tracing）、单细胞RNA测序（single-cell RNA sequencing）、光遗传学（optogenetics）与生理学实验，解析了小鼠心脏副交感调控环路。研究发现，脑干疑核（nucleus ambiguus, Amb）内的心脏支配神经元可分为两种分子特征、解剖结构与功能特性均显著不同的亚型。第一种亚型被命名为疑核心血管（ambiguus cardiovascular, ACV）神经元（每个疑核约含35个该类神经元），构成经典的心脏副交感环路：它们选择性支配心脏副交感神经节神经元的一个亚群，介导压力感受性反射，在血压升高时减慢心率并延缓房室结传导。第二种亚型为疑核心肺（ambiguus cardiopulmonary, ACP）神经元（每个疑核约含15个该类神经元），其支配的心脏神经节神经元与ACV神经元支配的神经元相互混杂且功能难以区分。此外，ACP神经元还支配大部分或全部肺脏副交感神经节神经元——克隆标记实验显示，单个ACP神经元可同时支配心脏与肺脏两个器官。ACP神经元介导潜水反射，即浸水后出现的心动过缓与支气管收缩同步反应。综上，心脏的副交感调控可分为两条平行环路：一条选择性调控心脏功能（ACV环路），另一条协同调控心脏与肺脏功能（ACP环路）。这一全新的心脏调控认知，可为心肺部疾病的治疗以及解析其他器官的调控与协同环路提供重要参考。本研究的单细胞mRNA表达谱源自通过心脏或喉部逆行标记的小鼠疑核神经元，测序时小鼠为出生后2至4日龄。互补DNA（complementary DNA, cDNA）通过Smart-seq2技术制备。