Neural Mechanisms Controlling Breathing In Mammals

该项目旨在提供有关哺乳动物呼吸运动产生和控制的基本神经机制的信息。长期的目标是解释呼吸运动的个体发育和神经发生的分子，生物物理，突触和网络特性的呼吸神经元在哺乳动物脑干和脊髓。目前的工作集中在脑干中产生呼吸节律的细胞和网络机制上。一系列相互关联的多学科研究正在进行中，以确定：（1）参与呼吸节律产生和传递的脑干网络的部位、细胞成分和结构;（2）形成呼吸振荡器的神经元的生物物理特性和突触相互作用;（3）节律调制和突触传递的神经化学机制;和（4）功能鉴定的神经元的分子特性。实验用来自胎儿、新生儿和幼年啮齿动物的离体脑干-脊髓和脑干切片制备物进行。以前，我们已经确定了关键的脑干位点（称为前Botzinger复合体）包含的神经元群体产生的节奏。我们进一步开发了新的方法，用于实时的结构和功能成像的节奏产生的神经元，以及神经元的节奏传输电路，利用红外和微分干涉对比（IR-DIC）成像与荧光成像的神经元标记的钙敏感染料同时进行。这种成像方法促进了呼吸网络神经元的识别，用于生物物理和突触特性的电生理研究以及神经元通道和受体表达的分子研究。通过这些方法，我们已经成像的活动和分析的生物物理特性的呼吸起搏神经元在前Botzinger复杂的体外，提供了最直接的实验证据，迄今为止，节奏的产生涉及一个网络的神经元专门的起搏特性。多光子成像的方法，将允许三维重建这个网络在前Botzinger复杂的目前正在开发中。对细胞膜生物物理特性的研究提供了额外的证据，表明持续的钠和钾泄漏电导代表了产生细胞起搏行为的关键离子电导机制。用RT-PCR对单个起搏细胞中表达的信使RNA进行分子分析，显示钠和钾通道的分布与持续钠和钾泄漏电导的重要作用一致。电生理学研究还表明，这些传导机制在呼吸节律的调节中起着关键作用，这些调节这些传导的神经化学物质包括5-羟色胺和P物质，以及生理控制信号包括二氧化碳和氧气。这些结果继续支持我们的混合起搏器网络模型，该模型是从以前的工作中制定的，以解释呼吸节律的产生和控制。计算方法已与实验研究并行使用，以对混合起搏器网络进行建模。我们的起搏神经元的生物药理学现实的计算模型得到了进一步的发展，并进行了新的调查，这些细胞的突触耦合群体的动态行为。这些模型的计算机模拟模拟了体外实验发现的单细胞和神经元群体活动的许多特征，包括非线性动力学现象产生的节律不稳定性，如起搏细胞网络中出现的准周期性。基于计算机的方法也得到了进一步的改进，以产生这些模拟的动画，允许可视化模型神经元的动态行为及其网络交互。这些模型目前正被应用于进一步探索和可视化神经系统发育不同阶段呼吸振荡器的操作原理。

This project is designed to provide information on basic neural mechanisms involved in the generation and control of respiratory movements in mammals. The long-range goal is to explain the ontogeny and neurogenesis of respiratory movements in terms of the molecular, biophysical, synaptic, and network properties of respiratory neurons in the mammalian brainstem and spinal cord. Current work focuses on cellular and network mechanisms generating the respiratory rhythm in the brainstem. A set of interrelated, multidisciplinary studies are ongoing to determine: (1) sites, cellular components, and architecture of brainstem networks involved in generation and transmission of respiratory rhythm; (2) biophysical properties and synaptic interactions of neurons forming the respiratory oscillator; (3) neurochemical mechanisms for modulation and synaptic transmission of rhythm; and (4) molecular properties of functionally identified neurons. Experiments are performed with isolated in vitro brainstem-spinal cord and brainstem slice preparations from fetal, neonatal, and juvenile rodents. Previously we have identified the critical brainstem locus (called the pre-Botzinger complex) containing the populations of neurons generating the rhythm. We have further developed novel methods for real-time structural and functional imaging of the rhythm-generating neurons, as well as neurons in rhythm-transmission circuits, utilizing infrared and differential interference contrast (IR-DIC) imaging performed simultaneously with fluorescence imaging of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of respiratory network neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of neuron channel and receptor expression. With these approaches, we have imaged the activity and analyzed biophysical properties of respiratory pacemaker neurons in the pre-Botzinger complex in vitro, providing the most direct experimental evidence to date that rhythm generation involves a network of neurons with specialized pacemaker properties. Methods for multi-photon imaging that will allow three-dimensional reconstruction of this network in the pre-Botzinger complex are currently under development. Studies of cellular membrane biophysical properties have provided additional evidence that persistent sodium and potassium leak conductances represent critical ionic conductance mechanisms generating cellular pacemaker behavior. Molecular profiling with RT-PCR of messenger RNA expressed in single pacemaker cells shows a profile of sodium and potassium channels consistent with an important role of persistent sodium and potassium leak conductances. Electrophysiological studies have also demonstrated that these conductance mechanisms are critically involved in the regulation of the breathing rhythm by a diverse set of neurochemicals that modulate these conductances, including serotonin and substance P, as well as physiological control signals including carbon dioxide and oxygen. These results continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of the breathing rhythm. Computational approaches have been used in parallel to experimental studies to model the hybrid pacemaker-network. Our biophysically realistic computational models of pacemaker neurons have been further developed and novel investigations were conducted on the dynamic behavior of synaptically coupled populations of these cells. Computer simulations with these models mimic many features of the single-cell and neuron population activity found experimentally in vitro, including instabilities of the rhythm produced by nonlinear dynamic phenomena such as quasiperiodicity arising in networks of pacemaker cells. Computer-based methods have also been further refined to produce animations of these simulations, allowing visualization of the dynamic behavior of the model neurons and their network interactions. These models are currently being applied to further explore and visualize principles of operation of the respiratory oscillator at different stages of nervous system development.