Neurophysiology of Breathing Behavior in Mice

 描述(申请人提供)：这个R01更新项目旨在从细胞和突触水平解释呼吸行为的神经起源。它促进了对脑干前Bötzinger复合体(PrebötC)的理解，该复合体被认为是迄今为止研究过的人类和所有陆地哺乳动物呼吸的主要部位。此外，该项目还研究了与PrebötC相邻的中间网状结构的中间神经元，这些中间神经元可能会产生呼吸运动前神经元。这个项目的智力驱动力是PI的团队和法国同事的发现，即围产期小鼠关键的导致节律的PrebötC中间神经元来自表达转录因子Dbx1的胚胎前体细胞(即Dbx1 PrebötC神经元)。该项目利用这一新知识，通过将Dbx1 CRE驱动小鼠与六个不同的FLOX停止报告菌株偶联来执行一系列体内和体外实验，如膜片钳记录、具有生理监测的细胞特异性激光消融，以及询问网络属性的光遗传操作。目的1用幼年和成年小鼠(体内和体外)检测Dbx1 PrebötC神经元在胚胎和新生儿发育阶段之后是否具有节律性。目的2利用体外胚胎和新生小鼠，结合细胞特异性激光消融方法，测试具有阵发性起搏器特性的PrebötC神经元是否对呼吸节律的产生是必需的，为解决24年来关于起搏器驱动的PrebötC节律的一个悬而未决的问题提供了一种新的途径。目的3利用体外培养的围产期小鼠，研究Dbx1神经元之间的突触联系并量化其输入输出关系。这些实验阐明了Dbx1 PrebötC神经元中反复出现的突触兴奋，这也是可能的节律性兴奋。目的利用围产期成年小鼠(体内和体外)，观察邻近中间网状结构中的Dbx1神经元是否作为舌头和咽部呼吸运动的第一层前运动神经元。呼吸控制回路的功能障碍会导致严重的健康问题，包括阻塞性和中枢性呼吸暂停，以及呼吸衰竭和死亡。这些疾病折磨着早产儿、儿童、成年人和神经退行性疾病患者。该项目具有重要意义，因为它描述了激活呼吸振荡器核心的关键遗传类别神经元(即Dbx1)的细胞和突触机制，这代表着我们在理解方面的革命性进展，将为抗击呼吸道病理的新的预防和治疗策略提供信息。PI是这项工作的理想科学家，因为他在呼吸神经生物学领域的领先记录，他与法国同事一起首次确定了Dbx1神经元在PrebötC中的作用，现在准备进一步发现它们的详细特性和下游的运动前驱对应物。如果这个项目成功，神经科学将最终知道哺乳动物重要的中央模式产生电路的细胞和突触起源，以及重要行为--呼吸的起源。

 DESCRIPTION (provided by applicant): This R01 renewal project aims to explain the neural origins of breathing behavior at the cellular and synaptic level. It advances understanding of the brainstem pre-Bötzinger complex (preBötC), which is acknowledged to be the principal site driving respiration in humans and all terrestrial mammals so far studied. Also, this project examines interneurons of the intermediate reticular formation, adjacent to the preBötC, which may give rise to respiratory premotor neurons. The intellectual driving force for this project is te discovery by the PI's team - and French colleagues - that the key rhythmogenic preBötC interneurons in perinatal mice are derived from embryonic precursors that express transcription factor Dbx1 (i.e., Dbx1 preBötC neurons). This project exploits this new knowledge and by coupling Dbx1 Cre-driver mice with six different flox-STOP reporter strains to perform a spectrum of experiments in vivo and in vitro such as patch-clamp recordings, cell-specific laser ablations with physiological monitoring, and optogenetic manipulations that interrogate network properties. Aim 1 uses juvenile and adult mice (in vivo and in vitro) to examine whether Dbx1 preBötC neurons are rhythmogenic beyond embryonic and neonatal stages of development. Aim 2 uses embryonic and neonatal mice in vitro in conjunction with cell-specific laser ablation methods to test whether preBötC neurons with bursting-pacemaker properties are obligatory for respiratory rhythm generation, offering a fresh approach to a 24-year-old unsolved problem regarding `pacemaker' driven preBötC rhythms. Aim 3 uses perinatal mice in vitro to characterize synaptic interconnections among Dbx1 neurons and quantify the input-output relationship. These experiments elucidate recurrent synaptic excitation in Dbx1 preBötC neurons, which is also putatively rhythmogenic. Aim 4 uses perinatal through adult mice (in vivo and in vitro) to examine whether Dbx1 neurons in the adjacent intermediate reticular formation serve as the first layer of premotor neurons for respiratory movements of the tongue (genioglossus) and pharynx. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project is significant because it characterizes the cellular and synaptic mechanisms that animate the key genetic class of neurons (i.e., Dbx1) at the core of the respiratory oscillator, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who - with French colleagues - first characterized the role of Dbx1 neurons in the preBötC and now is poised to further discover their detailed properties and downstream premotor counterparts. If this project succeeds, neuroscience would finally know the cellular and synaptic origins of a significant central pattern- generating circuit in a mammal and the point of origin for an important behavior, breathing.