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：(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特性和突触相互作用；(3)节律调节和突触传递的神经化学机制；(4)功能鉴定神经元的分子特性。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。

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 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 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 neurons with specialized pacemaker properties. Studies of membrane biophysical properties have identified a persistent sodium conductance as a candidate ionic conductance mechanism generating cellular pacemaker behavior. Molecular profiling with RT-PCR of messenger RNA expressed in single pacemaker cells shows a profile of voltage-activated sodium channels consistent with an important role of a persistent sodium conductance. 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 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.