Cellular and Molecular Identification of the Breathing Pacemaker Neurons

项目摘要 生命中有两个至关重要的起搏器：心脏起搏器和呼吸起搏器， preBötC（preBötC）。preBötC是脑干中约3000个神经元的集群， 周期性活动，每次活动爆发都会引发呼吸。与心脏起搏器相比， 呼吸节律产生的分子和细胞基础仍然未知，与呼吸节律相关的疾病也是如此。 它，如中枢性睡眠呼吸暂停和婴儿猝死。preBötC节律生成的流行模型， 称为“群起搏器”模型，提出每次呼吸都是由一个紧急的preBötC网络触发的 现象。这个模型的一个重要假设是没有专门的呼吸起始神经元。 然而，根据观察到的各种preBötC神经元放电模式，包括在 每次呼吸（吸气前），以及前BötC神经元I的意想不到的分子和功能多样性 在我读博士时发现的我假设，在心脏中，有特定的神经元启动每个 呼吸，呼吸起搏神经元，并建议在这项研究中识别和表征它们 提议作为加州大学旧金山分校桑德勒研究员和早期独立奖的获得者，我计划首先 通过单细胞基因表达分析全面绘制preBötC细胞类型， 呼吸起搏神经元通过它们表达对心脏起搏重要的相同离子通道。 此外，我计划通过呼吸期间的预期活动来识别候选的呼吸启动神经元 （吸气前）和它们在体外的自主节律活动（起搏器活动）。最后，我将确定 我在博士学位时发现了大约175个preBötC神经元，的 接收呼吸起搏器活动。我预测，这三种独立的方法将在 同样的preBötC亚型，假定的呼吸起搏神经元，然后我将使用交叉遗传学方法， 测试所识别的神经元是否具有呼吸起搏器特性的策略：自主节律活动， 吸气前活动、启动呼吸的能力和呼吸要求。分子和功能 识别呼吸起搏神经元将是一个变革性的发现，最终导致 呼吸节律和心律失常是婴儿中最致命的疾病之一， 生成的.这种对呼吸节律产生的机械性理解将提供一种发展 控制通气的药理学方法，这将影响多个医学领域，特别是 生命科学和重症监护医学。在我最近的博士学位。我已经证明了非凡的分子 preBötC内的多样性，并证明了少量分子上不同的preBötC细胞类型， 在呼吸行为中具有高度特异性的功能。我准备继续解剖 目的是识别启动呼吸和控制呼吸节奏的核心神经元。

Project Summary There are two critical pacemakers for life: the cardiac pacemaker and the breathing pacemaker, the preBötzinger Complex (preBötC). The preBötC is a cluster of ~3000 neurons in the brainstem that are cyclically active, with each burst of activity initiating a breath. In contrast to the cardiac pacemaker, the molecular and cellular basis of breathing rhythm generation remains unknown, as do diseases associated with it, such as central sleep apnea and sudden infant death. The prevailing model of preBötC rhythm generation, called the `group-pacemaker' model, proposes that each breath is triggered by an emergent preBötC network phenomena. An important assumption of this model is that there are not dedicated breath-initiating neurons. However, based on the observed variety of preBötC neuron firing patterns, including ones that fire just before each breath (pre-inspiratory), and the unexpected molecular and functional diversity of the preBötC neurons I discovered during my Ph.D., I hypothesize that, as in the heart, there are specific neurons that initiate each breath, breathing pacemaker neurons, and propose to identify and characterize them in this research proposal. As a UCSF Sandler Fellow and recipient of the Early Independence Award, I plan to first comprehensively map preBötC cell types with single cell gene expression analysis and identify candidate breathing pacemaker neurons by their expression of the same ion channels important for cardiac pacemaking. Additionally, I plan identify candidate breath-initiating neurons by their anticipated activity during breathing (pre-inspiratory) and their autonomous, rhythmic activity in vitro (pacemaker activity). Lastly, I will identify candidate pacemakers by their proposed connectivity to ~175 preBötC neurons I identified in my Ph.D. that receive breathing pacemaker activity. I predict that these three independent approaches will converge on the same preBötC subtypes, the presumed breathing pacemaker neurons and I will then use intersectional genetic strategies to test if the identified neurons have breathing pacemaker properties: autonomous rhythmic activity, pre-inspiratory activity, ability to initiate a breath, and requirement for breathing. The molecular and functional identification of respiratory pacemaker neurons will be a transformative discovery, leading to the eventual resolution of how respiratory rhythms and arrhythmias, some of the most deadly diseases in infants, are generated. This mechanistic understanding of breathing rhythm generation will provide an avenue to develop pharmacological approaches to control ventilation, which would impact multiple medical fields, especially neonatology and critical care medicine. In my recent Ph.D. work, I have demonstrated extraordinary molecular diversity within the preBötC and demonstrated that small numbers of molecularly distinct preBötC cell types have highly specific functions in the breathing behavior. I am poised to continue this dissection with the objective of identifying the core neurons that initiate a breath and control the pace of breathing.