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；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；以及节律调节和突触传递的神经化学机制。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。包含产生节律的神经元群体的关键脑干基因已经被识别出来。开发了新的方法在保留功能活跃的呼吸网络的延髓薄片中分离该基因座(称为Pre-Botzinger复合体)，从而允许在细胞和网络水平上同时进行机制的实验分析。我们进一步开发了新的方法，利用红外照明和差分干涉对比(IR-DIC)光学技术对节律产生神经元进行实时结构和功能成像，同时对钙敏感染料标记的神经元进行荧光成像。这种成像方法有助于识别产生节律的神经元，用于生物物理和突触特性的电生理学研究。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，提供了迄今为止最直接的实验证据，证明节律产生涉及具有特殊起搏器特性的神经元。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，以解释节律的产生。计算方法已与实验研究并行使用，以模拟呼吸神经元和网络。我们对起搏器神经元的生物物理现实计算模型进行了改进，并对这些细胞突触耦合群体的动态行为进行了新的研究。使用这些模型的模拟模拟了在体外实验中发现的神经元活动和突触相互作用的许多特征。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呼吸振荡器在神经系统发育的不同阶段的工作原理。-呼吸、哺乳动物、中枢神经系统、脑干和脊髓、神经成像、神经振荡器

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; and neurochemical mechanisms for modulation and synaptic transmission of rhythm. Experiments are performed with isolated in vitro brainstem-spinal cord and brainstem slice preparations from fetal, neonatal, and juvenile rodents. The critical brainstem locus containing the populations of neurons generating the rhythm has been identified. Novel methods were developed to isolate this locus (called the pre-Botzinger complex) in thin slices of the medulla which retain functionally active respiratory networks, 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 utilizing infrared illumination and differential interference contrast (IR-DIC) optics simultaneously with fluorescence imaging of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of the rhythm generating neurons for electrophysiological studies of biophysical and synaptic properties. With these approaches, we have imaged the activity 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 which was formulated from previous work to explain rhythm generation. Computational approaches have been used in parallel to experimental studies to model respiratory neurons and networks. Our biophysically realistic computational models of the pacemaker neurons have been refined and novel investigations were conducted on the dynamic behavior of synaptically-coupled populations of these cells. Simulations with these models mimic many features of the neuron activity and synaptic interactions 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. - respiration, mammals, central nervous system, brainstem and spinal cord, neural imaging, neural oscillators