Neurophysiology of breathing behavior in neonatal mice in vitro

描述(申请人提供)：这个R01项目将促进我们对在人类和所有哺乳动物中产生和控制呼吸行为的脑干神经回路的理解。呼吸是心肺生理学不可分割的一部分，了解其神经起源对人类健康具有重要意义。有节奏的呼吸运动开始于胚胎发育期间，由脑干呼吸神经元的协调活动发出。节律产生神经元的一个关键群体包含在一个名为Preb“tzinger Complex(Preb”tC)的位置。Preb“tC的发现使许多可以在体外进行的强大实验成为可能，并使我们对呼吸的神经生理学有了当代的理解。然而，关键的问题仍然没有答案。鉴于Preb”tC中与呼吸相关的和非呼吸的神经元的异质性，我们能否发现哪些神经元是关键的节律生成器？如果可以识别节律神经元(我们认为确实可以)，那么我们能确定节律产生的细胞、突触和分子水平的机制吗？最后，在过去的十年中，多肽能调节呼吸节律的重要性已经得到了广泛的认识，但其潜在的生物物理机制仍然不完全清楚。本项目通过研究体外脑干薄片制备中的Preb“tC来寻求这些具体问题的答案。特异性AIM 1将评估Preb”tC的细胞组成。转基因小鼠模型将被用来将荧光标签应用于遗传上不同的亚群，然后选择性和连续地损伤它们，以测试它们在节律发生中各自的作用。特定的AIM 2将研究产生吸气相关猝发的突触-树突活性膜特性。特定的AIM 3将研究突触前兴奋性传递的抑制是否有助于猝发终止。特定的AIM 4旨在通过检测突触后膜的属性来补充特定的AIM 3，突触后膜也起到终止吸气爆发的作用。最后，特定的AIM 5将确定关键神经肽(和其他神经信使)调节呼吸的离子通道。在这个项目中获得的新知识将有助于治疗和预防因大脑和中枢神经系统故障而导致的呼吸障碍。此外，在体外受控条件下研究可测量的行为，如呼吸，有助于揭示将神经元、突触和分子与全面的生理行为联系起来的重要原理，这将是神经科学和心肺生理学的极大兴趣。

DESCRIPTION (provided by applicant): This R01 project will advance our understanding of the brainstem neural circuits that generate and control breathing behavior in humans and all mammals. Breathing is an integral part of cardiopulmonary physiology and understanding its neural origins has significant implications for human health. Rhythmic breathing movements begin during embryonic development and emanate from coordinated activity in brainstem respiratory neurons. One key population of rhythm-generating neurons is contained in a site called the preB"tzinger complex (preB"tC). The discovery of the preB"tC made possible many powerful experiments that could be performed in vitro, and led to our contemporary understanding of the neurophysiology of respiration. Nevertheless, critical questions remain unanswered. Given the heterogeneity of respiratory-related and non-respiratory neurons in the preB"tC, can we discover which neurons are the key rhythm generators? If rhythmogenic neurons can be identified (and we argue that indeed they can), then can we ascertain the cellular, synaptic, and molecular-level mechanisms that underlie rhythm generation? Finally, the importance of peptidergic modulation of respiratory rhythm has been widely recognized in the past decade, but its underlying biophysical mechanisms remain incompletely understood. This project seeks answers to these specific questions by studying the preB"tC in thin brainstem slice preparations in vitro. SPECIFIC AIM 1 will evaluate the cellular composition of the preB"tC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct sub-populations, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. SPECIFIC AIM 2 will examine the synaptic-dendritic active membrane properties that generate inspiratory-related bursts. SPECIFIC AIM 3 will investigate whether presynaptic depression of excitatory transmission contributes to burst termination. SPECIFIC AIM 4 is designed to complement SPECIFIC AIM 3 by examining the postsynaptic membrane properties that also act to terminate inspiratory bursts. Finally, SPECIFIC AIM 5 will determine the ion channels that underlie respiratory modulation by key neuropeptides (and other neuromessengers). The new knowledge acquired during this project will aid in the treatment and prophylaxis of breathing disorders that result from failures in the brain and central nervous system. Moreover, studying a measurable behavior like breathing under controlled in vitro conditions helps reveal important principles that link neurons, synapses, and molecules to full-scale physiological behaviors, which will be of great interest in neuroscience as well as cardiopulmonary physiology.