Neural Mechanisms Controlling Breathing In Mammals

研究重点是啮齿动物脑干中产生呼吸节律和神经活动模式的细胞和网络机制。 本实验采用新生和幼年大鼠离体脑干-脊髓原位灌流标本和离体脑干切片标本进行实验研究。以前，我们已经确定了脑干基因座（称为前Botzinger复杂）包含参与节律产生的神经元群体。我们进一步开发了新的方法，这些神经元的实时结构和功能成像，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）成像与荧光成像的神经元标记的钙敏感染料同时进行。 这种成像方法有利于识别呼吸网络神经元的生物物理和突触特性的电生理研究，以及神经元通道和受体表达的分子研究。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神经元在新生啮齿动物前Botzinger复杂的体外，提供了最直接的实验证据，迄今为止，节奏的产生涉及一个网络的神经元与专门的细胞特性。多光子成像的方法，将允许三维重建这个网络在前Botzinger复杂的目前正在开发中。对细胞膜生物物理特性的研究提供了额外的证据，表明持续的钠和钾泄漏电导代表了节律产生的关键离子电导机制。在单个功能鉴定的神经元中表达的信使RNA的RT-PCR分子分析显示钠和钾通道的概况与持续的钠和钾泄漏电导的重要作用一致。电生理学研究还表明，这些传导机制在呼吸节律的调节中起着关键作用，这些调节这些传导的神经化学物质包括5-羟色胺和P物质，以及生理控制信号包括二氧化碳和氧气。用更完整的脑干-脊髓原位制备物进行的电生理学研究证实了这些细胞和网络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，以解释完整的哺乳动物神经系统中呼吸节律的产生和控制。新的计算方法，包括脑干神经网络的大规模建模已被用于并行实验研究。我们的呼吸神经元的生物药理学现实的计算模型得到了进一步的发展，并进行了新的调查，这些网络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和原位实验发现的单细胞和神经元群体活动的许多特征，包括非线性动力学现象产生的节律不稳定性，如网络中出现的准周期性。这些模型目前正被应用于进一步探索脑干呼吸网络在神经系统发育的不同阶段的运作原理。

Research focused on cellular and network 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 juvenile 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 the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of respiratory network 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 in vitro, providing the most direct experimental evidence to date that rhythm generation involves a network of neurons with specialized cellular properties. 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 rhythm generation. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons shows a profile of sodium and potassium channels consistent with an important role of persistent sodium and potassium leak conductances. Electrophysiological studies have also demonstrated that these conductance mechanisms are critically involved in the regulation of the breathing rhythm 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 network 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 the breathing rhythm 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. These models are currently being applied to further explore principles of operation of brainstem respiratory networks at different stages of nervous system development.