Neurophysiology of Breathing Behavior in Mice

 描述（由申请人提供）：这个R 01更新项目旨在解释细胞和突触水平上呼吸行为的神经起源。它推进了对脑干前Bötinger复合体（preBötC）的理解，该复合体被认为是迄今为止研究的人类和所有陆生哺乳动物驱动呼吸的主要部位。此外，该项目还检查了中间网状结构的中间神经元，邻近preBötC，这可能会产生呼吸前运动神经元。该项目的智力驱动力是PI团队和法国同事的发现，即围产期小鼠中关键的节律性preBötC中间神经元来源于表达转录因子Dbx 1的胚胎前体（即，Dbx 1 preBötC神经元）。该项目利用了这一新知识，并通过将Dbx 1 Cre-driver小鼠与六种不同的EST-STOP报告菌株偶联，在体内和体外进行了一系列实验，如膜片钳记录，细胞特异性激光消融与生理监测，以及询问网络特性的光遗传学操作。目的1使用幼年和成年小鼠（体内和体外）来研究Dbx 1 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使用胚胎和新生小鼠在体外结合细胞特异性激光消融方法，以测试是否preBötC神经元与爆发起搏器的属性是强制性的呼吸节律的产生，提供了一个新的方法来解决一个24岁的未解决的问题，关于'起搏器'驱动的preBötC节奏。目的3利用围产期小鼠在体外Dbx 1神经元之间的突触互连和量化的输入输出关系。这些实验阐明了Dbx 1 preBötC神经元中反复出现的突触兴奋，这也是puerythromogenic。目的4使用围产期通过成年小鼠（在体内和体外），以检查是否Dbx 1神经元在相邻的中间网状结构作为第一层的前运动神经元的呼吸运动的舌头（颏舌肌）和咽。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。这些病症折磨早产儿、儿童、成人和患有神经退行性疾病的患者。这个项目是重要的，因为它的特点细胞和突触机制，动画的关键遗传类神经元（即，Dbx 1）在呼吸振荡器的核心，这代表了一个变革性的进步，我们的理解，将告知新的预防和治疗策略，以打击呼吸病理。PI是这项工作的理想科学家，因为他是呼吸神经生物学的领导者，他与法国同事一起首次描述了Dbx 1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运动前区对应物。如果这个项目成功，神经科学将最终知道哺乳动物中一个重要的中枢模式生成回路的细胞和突触起源，以及呼吸这一重要行为的起源点。

 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.