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。长期目标是从哺乳动物脑干和脊髓中呼吸神经元的分子、生物物理、突触和网络特性来解释呼吸运动的个体发育和神经发生。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位置、细胞成分和结构；(2)形成呼吸振荡器的神经元的生物物理性质和突触相互作用；(3)节律调节和突触传递的神经化学机制；以及(4)功能识别神经元的分子特性。实验是用分离的体外脑干-脊髓和脑干切片标本从胎儿、新生儿和幼年啮齿动物身上进行的。在此之前，我们已经确定了包含产生节律的神经元群体的关键脑干基因(称为前波辛格复合体)。我们进一步发展了新的方法，利用红外和差分干涉对比(IR-DIC)成像与钙敏感染料标记的神经元的荧光成像，对节律产生神经元以及节律传递电路中的神经元进行实时结构和功能成像。这种成像方法有助于识别呼吸网络神经元，用于生物物理和突触性质的电生理学研究，以及神经元通道和受体表达的分子研究。通过这些方法，我们已经在体外对前Botzinger复合体中呼吸起搏器神经元的活动进行了成像和生物物理性质的分析，提供了迄今为止最直接的实验证据，证明节律的产生涉及具有特殊起搏器特性的神经元网络。目前正在开发多光子成像方法，以便能够在前波辛格复合体中对这一网络进行三维重建。对细胞膜生物物理性质的研究提供了更多的证据，表明持续的钠和钾泄漏电导是导致细胞起搏器行为的关键离子电导机制。在单个起搏细胞中表达的信使RNA的RT-PCR分子图谱显示，钠和钾通道的轮廓与持续的钠和钾泄漏电导的重要作用一致。电生理学研究还表明，这些电导机制与调节呼吸节律的多种神经化学物质密切相关，这些神经化学物质包括5-羟色胺和P物质，以及包括二氧化碳和氧气在内的生理控制信号。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形成的，用于解释呼吸节奏的产生和控制。计算方法已与实验研究并行使用，以模拟混合起搏器网络。我们进一步发展了起搏神经元的生物物理现实计算模型，并对这些细胞突触耦合群体的动态行为进行了新的研究。使用这些模型的计算机模拟模拟了在体外实验中发现的单细胞和神经元群体活动的许多特征，包括由非线性动态现象产生的节律的不稳定性，例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呼吸振荡器在神经系统发育的不同阶段的工作原理。

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.