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生和神经发生。目前的工作主要集中在脑干中产生呼吸节律的细胞和网络机制。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特性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。实验采用胚胎、新生儿和幼年啮齿动物的离体脑干-脊髓和脑干切片制备。先前我们已经确定了关键的脑干位点，其中包含产生节律的神经元群。新的方法被进一步开发出来，在保留功能活跃的呼吸网络的髓质薄片中分离出这个位点（称为前botzinger复合体），在前botzinger复合体“岛”中，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，用于实时结构和功能成像的节奏产生神经元，以及在节奏传输电路中的神经元，利用红外照明和微分干涉对比（IR-DIC）光学与钙敏感染料标记神经元的荧光成像同时进行。这种成像方法有助于识别节律产生/传递回路神经元，用于生物物理和突触特性的电生理研究。通过这些方法，我们在体外对pre-Botzinger复合体中呼吸起搏器神经元的活性进行了成像并分析了其生物物理特性，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，该模型是根据之前的工作制定的，用于解释心律产生。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计算模型进行了进一步的开发，并对这些细胞的突触偶联群体的动态行为进行了新的研究。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。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，从而使模型神经元的动态行为及其网络相互作用可视化。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不同阶段的操作原理。

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; 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. 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 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/transmission-circuit neurons for electrophysiological studies of biophysical and synaptic properties. 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.