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。实验采用分离的、原位灌流的脑干-脊髓和体外制备的新生和幼年啮齿动物脑干切片。在此之前，我们已经确定了包含参与节律产生的神经元群体的脑干基因座(称为前波辛格复合体)。我们进一步开发了对这些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的新方法，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同时进行。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。用这些方法，我们已经在体外对新生啮齿动物前Botzinger复合体中呼吸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细胞属性的神经元网络。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节律产生的关键离子电导机制。用RT-PCR对单个功能鉴定神经元中表达的信使RNA进行分子图谱分析表明，钠和钾通道的分布与持续的钠和钾泄漏电导的重要作用相一致。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。以更完整的脑干-脊髓原位标本进行的电生理学研究证实了这些细胞和网络机制在新生儿和成熟哺乳动物神经系统中的重要性。这些结果继续支持我们从以前的工作中提出的混合起搏器网络模型，该模型解释了完整哺乳动物神经系统中呼吸节奏的产生和控制。新的计算方法，包括脑干神经网络的大规模建模，已经被用于平行于实验研究。我们进一步发展了呼吸神经元的生物物理现实计算模型，并对这些网络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和现场实验中发现的单细胞和神经元群体活动的许多特征，包括由非线性动力学现象产生的节律的不稳定性，例如网络中出现的准周期。这些模型目前正被应用于进一步探索神经系统发育不同阶段脑干呼吸网络的运行原理。

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 situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal and juvenile rodents. Previously we have identified the brainstem locus (called the pre-Botzinger complex) containing populations of neurons participating in rhythm generation. We have further developed novel methods for real-time structural and functional imaging of these 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 neurons in the neonatal rodent pre-Botzinger complex in vitro, providing the most direct experimental evidence to date that rhythm generation involves a network of neurons with specialized cellular 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 for rhythm generation. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons 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. Electrophysiological studies performed with more intact preparations of the brainstem-spinal cord in situ have confirmed the importance of these cellular and network mechanisms in both the neonatal and mature mammalian nervous systems. 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 in the intact mammalian nervous system. Novel computational approaches including large-scale modeling of brainstem neural networks have been used in parallel to experimental studies. Our biophysically realistic computational models of respiratory neurons have been further developed and novel investigations were conducted on the dynamic behavior of synaptically coupled populations of these network cells. Computer simulations with these models mimic many features of the single-cell and neuron population activity found experimentally in vitro and in situ, including instabilities of the rhythm produced by nonlinear dynamic phenomena such as quasiperiodicity arising in networks. These models are currently being applied to further explore principles of operation of brainstem respiratory networks at different stages of nervous system development.