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。一组相互关联的，多学科的研究正在进行中，以确定：网站，细胞成分，并参与呼吸节律的产生和传输的脑干网络的架构;生物物理特性和神经元形成的呼吸振荡器的突触相互作用;神经化学机制的调制和突触传递的节奏;和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包含的神经元群体产生的节奏。新的方法被进一步开发，以隔离该位点（称为前Botzinger复合体）在薄切片的髓质保留功能活跃的呼吸网络，并在前Botzinger复合体“岛”，允许实验分析的机制，同时在细胞和网络水平。我们已经进一步开发了新的方法，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）成像的同时与荧光成像的神经元标记的钙敏感染料。这种成像方法有利于识别的节奏产生/传输电路神经元的生物物理和突触特性的电生理研究，以及神经元通道表达的分子研究。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，提供最直接的实验证据，迄今为止，节奏的产生涉及神经元专门的起搏特性。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，以解释节律的产生。计算方法已被用于并行实验研究的混合起搏器网络模型。我们的起搏神经元的生物药理学现实的计算模型已得到进一步发展，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。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: sites, cellular components, and architecture of brainstem networks involved in generation and transmission of respiratory rhythm; biophysical properties and synaptic interactions of neurons forming the respiratory oscillator; neurochemical mechanisms for modulation and synaptic transmission of rhythm; and 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 containing the populations of neurons generating the rhythm. Novel methods were further developed to isolate this locus (called the pre-Botzinger complex) in thin slices of the medulla which retain functionally active respiratory networks, and in pre-Botzinger complex "islands", allowing experimental analysis of mechanisms concurrently at cellular and network levels. 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 simultaneously with fluorescence imaging of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of the rhythm-generating/transmission-circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of neuron channel 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 neurons with specialized pacemaker properties. These results continue to support our hybrid pacemaker-network model that was formulated from previous work to explain rhythm generation. Computational approaches have been used in parallel to experimental studies to model the hybrid pacemaker-network. Our biophysically realistic computational models of the 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. 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.