Neural Mechanisms Controlling Breathing In Mammals

控制哺乳动物呼吸的神经机制

• 批准号: 6990663
• 负责人: Jeffrey c Smith
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6990663
• 关键词: Rodentias; bioperiodicity; brain stem; computational neuroscience; computer simulation; developmental neurobiology; method development; neural information processing; neural transmission; neurochemistry; neuroimaging; neurons; neurophysiology; neuroregulation; polymerase chain reaction; potassium channel; pulmonary respiration; sodium channel; synapses; three dimensional imaging /topography

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.

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。远程目标是根据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。当前的工作着重于在脑干中产生呼吸节奏的细胞和网络机制。正在进行一系列相互关联的多学科研究以确定：（1）参与呼吸节奏的产生和传播的脑干网络的位点，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）节奏调节和突触传播的神经化学机制； （4）功能鉴定的神经元的分子特性。实验是用胎儿，新生儿和青少年啮齿动物的分离的体外脑茎脊髓和脑干切片制剂进行的。以前，我们已经确定了包含产生节奏的神经元种群的关键脑干基因座（称为前杂化络合物）。我们进一步开发了用于节奏生成神经元的实时结构和功能成像的新颖方法，以及节奏传递循环中的神经元，利用红外和不同的干扰对比（IR-DIC）成像，同时执行的神经元与钙敏感粉的神经元同时进行。这种成像方法促进了呼吸网络神经元的鉴定，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，我们已经成像了活性，并分析了在体外肉毒前复合体中呼吸性起搏器神经元的生物物理特性，这提供了迄今为止最直接的实验证据，即节奏生成涉及具有专门的起搏器特性的神经元网络。当前正在开发中，可以在前博客综合体中进行三维重建的多光子成像方法。细胞膜生物物理特性的研究提供了其他证据，表明持续的钠和钾泄漏电导代表了产生细胞过心器行为的关键离子电导机制。用单个起搏器细胞中表达的Messenger RNA的RT-PCR分子分析显示出与持续性钠和钾泄漏电导的重要作用一致的钠和钾通道的轮廓。电生理研究还表明，这些电导机制与一组调节这些电导的多种神经化学物质（包括5-羟色胺和物质P）以及包括二氧化碳和氧气在内的生理控制信号进行了调节，这些电导机制严重参与了呼吸节奏的调节。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提出的，以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。我们对起搏器神经元的生物物理现实计算模型已经进一步开发，并就这些细胞的突触耦合种群的动态行为进行了新的研究。这些模型的计算机模拟模仿了单细胞和神经元种群活性的许多特征，在体外实验发现，包括非线性动态现象产生的节奏的不稳定性，例如在Pacemaker细胞网络中产生的quasiperiodicitigity。基于计算机的方法还进一步完善了这些模拟的动画，从而可以可视化模型神经元的动态行为及其网络相互作用。这些模型目前正在应用于在神经系统发育的不同阶段进一步探索和可视化呼吸振荡器的操作原理。