Neuronal basis of ventilation in vertebrates

呼吸也许是所有脊椎动物行为中最重要、最有力、最有节奏的。脑干中的神经元回路产生呼吸节奏，并形成每次呼吸的运动模式，以满足代谢、生物力学和行为目标。然而，如何实现这一目标尚未解决。***我们通过关注分离的牛蛙脑干来解决这些问题。这种准备可以产生至少24小时类似于完整动物的呼吸运动模式。蝌蚪的这种弹性和发育性使青蛙成为教授神经科学和研究脊椎动物神经元发育和功能的理想选择。我们发现牛蛙和哺乳动物控制呼吸的神经回路有惊人的相似之处，尽管它们的呼吸机制存在根本差异。我们在分离的牛蛙脑干中发现了两个耦合的内源性节律性神经网络（即振荡器），一个用于口腔通气，另一个用于肺充气。哺乳动物的两个脑干振荡器也被提出，一个用于灵感，另一个用于死亡。在哺乳动物和牛蛙中假定的振子占据相似的菱形位置，并且似乎共享几个功能特性。我们最近的数据表明，青蛙脑干中存在第三个振荡器，即“启动振荡器”，它似乎是导致肺部膨胀之前严重的口腔收缩的原因。这个振子不同于颊振子，它横跨肺振子，占据更大的脑干区域。这些发现提出了重要的基本问题：振荡器范式如何很好地解释体内每次呼吸时发生的肌肉补充模式的复杂性（泵肌和气道阀的时间）？振子是如何连接的？其他呼吸空气的脊椎动物是否在每个通气阶段都有不同的振荡器？如果有，它们是如何进化的？***本研究的目的是通过批判性地评估振荡器结构作为脊椎动物呼吸节律产生的基本原理来扩大对呼吸回路的理解。在接下来的5年里，我和我的学员们将：***(1)使用标准的神经生物学技术在退化的牛蛙身上测试多振荡器范式，并确定振荡器是否单独解释了每次呼吸时泵肌和瓣膜激活的精确时间。***(2)记录和标记分离牛蛙脑干振荡器内的单个神经元以确定功能表型，并使用原位杂交检测定义哺乳动物呼吸节律产生神经元的分子标记的局部表达。***(3)使用原位杂交技术来标记青蛙和哺乳动物的振动子，以比较和对比其他低等脊椎动物的呼吸回路结构

Breathing is perhaps the most vital, robust and rhythmic of any vertebrate behavior. A neuronal circuit in the brainstem generates the respiratory rhythm and shapes the motor pattern of each breath to meet metabolic, biomechanical and behavioral goals. However, how this is achieved is not resolved. ***We have addressed these issues by focusing on the isolated bullfrog brainstem. This preparation produces breathing motor patterns resembling those of intact animals for at least 24 hrs. This resilience and the developmental accessibility of tadpoles makes frogs ideal for teaching neuroscience and for studying neuronal development and function in vertebrates.***We discovered striking similarities in the neuronal circuits controlling breathing between bullfrogs and mammals despite fundamental differences in their breathing mechanics. We found two coupled, endogenously rhythmogenic neural networks (i.e., oscillators) in the isolated bullfrog brainstem, one for buccal ventilation, the other for lung inflation. Two brainstem oscillators in mammals have also been proposed, one for inspiration, the other for expiration. The putative oscillators in mammal and bullfrog occupy similar rhombomeric locations and appear to share several functional properties. Our recent data suggest a third oscillator in the frog brainstem, the `priming oscillator' that appears responsible for the large buccal deflation that precedes lung inflation. This oscillator is distinct from the buccal oscillator and spans the lung oscillator occupying a much larger brainstem region. ***These findings raise important fundamental questions: How well does the oscillator paradigm account for the complexity of the muscle recruitment pattern that occurs during each breath (timing of pump muscles and airway valves) in vivo? How are the oscillators connected? Do other air-breathing vertebrates have distinct oscillators for each phase of ventilation, and if so, how did they evolve?***The objective of the proposed research is to expand understanding of breathing circuits by critically evaluating the oscillator construct as an underlying principle of respiratory rhythm generation in vertebrates. In the next 5 years, my trainees and I will:***(1) Use standard neurobiological techniques to test the multiple oscillator paradigm in the decerebrate bullfrog and determine if oscillators alone account for the precise timing of pump-muscles and valve activation that occurs with each breath.***(2) Record and label individual neurons within oscillators in isolated bullfrog brainstems to determine functional phenotypes, and use in situ hybridization to test for local expression of molecular markers that define mammalian respiratory rhythm generating neurons.***(3) Use in situ hybridization for markers that demarcate oscillators in frogs and mammals to compare and contrast the architecture of breathing circuits in other lower vertebrates.**