Neural Mechanisms Controlling Breathing In Mammals

研究集中在产生啮齿动物脑干呼吸节律和神经活动模式的细胞和电路机制上。分别用新生大鼠和成年大鼠分离、原位灌流的脑干-脊髓和体外脑干切片进行实验研究。在此之前，我们已经确定了包含参与节律产生的神经元群体的脑干基因座(称为前波辛格复合体)。我们进一步开发了对这些神经元以及节律传递电路中的神经元进行实时结构和功能成像的新方法，利用红外和差分干涉对比(IR-DIC)成像与钙敏感染料标记的神经元的活动模式的荧光成像同时进行。这种成像方法有助于识别呼吸回路神经元，用于生物物理和突触性质的电生理学研究，以及神经元通道和受体表达的分子研究。通过这些方法，我们已经在体外对新生啮齿动物前Botzinger复合体和节律传递电路中呼吸神经元的活动进行了成像和生物物理特性分析，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细胞属性的神经元网络，该网络赋予呼吸回路多种产生呼吸振荡的机制。目前正在开发多光子成像方法，以便能够在前波辛格复合体中对这一网络进行三维重建。对细胞膜生物物理性质的研究提供了更多的证据，表明持续的钠和钾泄漏电导是产生和控制呼吸振荡的关键离子电导机制。用RT-PCR对单个功能鉴定的神经元中表达的信使RNA进行分子图谱分析，以及免疫组织化学研究，表明钠、钾和神经递质受体连接的通道的图谱与持续性钠和漏电导的重要作用一致。电生理学研究还表明，这些电导机制与多种神经化学物质调节呼吸节律/模式有关，这些神经化学物质包括5-羟色胺和P物质，以及包括二氧化碳和氧气在内的生理控制信号。以更完整的脑干-脊髓原位标本进行的电生理学研究证实了这些细胞和回路机制在新生儿和成熟哺乳动物神经系统中的重要性。这些结果继续支持我们从以前的工作中提出的混合起搏器网络模型，该模型解释了完整哺乳动物神经系统中呼吸节律和模式的产生和控制。新的计算方法，包括脑干神经网络的大规模建模，已经被用于平行于实验研究。我们进一步发展了呼吸神经元的生物物理现实计算模型，并对这些网络细胞突触耦合群体的动态行为进行了新的研究。使用这些模型的计算机模拟模拟了在体外和现场实验中发现的单细胞和神经元群体活动的许多特征，包括由非线性动力学现象产生的节律的不稳定性，如网络中出现的准周期。呼吸神经控制系统的模型也已经发展起来，它将神经回路动力学与外周氧气和二氧化碳交换、血气运输以及中枢呼吸回路的生理反馈调节相结合。这些模型目前正被应用于进一步探索脑干呼吸回路在神经系统发育的不同阶段和不同(病理)生理条件下的工作原理。

Research focused on cellular and circuit mechanisms generating the respiratory rhythm and neural activity patterns in the brainstem of rodents. Experimental studies were performed with isolated in situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal and mature rats. Previously we have identified the brainstem locus (called the pre-Botzinger complex) containing populations of neurons participating in rhythm generation. We have further developed novel methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing infrared and differential interference contrast (IR-DIC) imaging performed simultaneously with fluorescence imaging of activity patterns of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of respiratory 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 neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro, providing the most direct experimental evidence to date that rhythm generation involves a network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. 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 for generation and control of respiratory oscillations. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons, as well as immunohistochemical studies, show a profile of sodium, potassium, and neurotransmitter receptor-linked channels consistent with an important role of persistent sodium and leak conductances. Electrophysiological studies have also demonstrated that these conductance mechanisms are critically involved in the regulation of the breathing rhythm/pattern 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. Electrophysiological studies performed with more intact preparations of the brainstem-spinal cord in situ have confirmed the importance of these cellular and circuit mechanisms in both the neonatal and mature mammalian nervous systems. These results continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratoy rhythm and pattern in the intact mammalian nervous system. Novel computational approaches including large-scale modeling of brainstem neural networks have been used in parallel to experimental studies. Our biophysically realistic computational models of respiratory neurons have been further developed and novel investigations were conducted on the dynamic behavior of synaptically coupled populations of these network cells. Computer simulations with these models mimicked many features of the single-cell and neuron population activity found experimentally in vitro and in situ, including instabilities of the rhythm produced by nonlinear dynamic phenomena such as quasiperiodicity arising in networks. Models of the respiratory neural control system that couple neural circuit dynamics with peripheral oxygen and carbon dioxide exchange, blood gas transport, and physiological feedback regulation of central respiratory circuits have also been developed. These models are currently being applied to further explore principles of operation of brainstem respiratory circuits at different stages of nervous system development and under various (patho)physiological conditions.