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电路机制。 本实验采用新生大鼠和成年大鼠离体脑干-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）成像与荧光成像的活动模式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回路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复杂和节奏传输电路在体外，提供最直接的实验证据，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，赋予呼吸电路与多种机制产生呼吸振荡。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。前Botzinger复合体中神经元突触相互作用和细胞膜生物物理特性的研究，包括先进的电生理方法，如“动态钳”，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的神经节律和模式的产生和控制。这些研究提供了额外的证据表明，神经元持续钠电流和钾泄漏电导的产生和控制的呼吸振荡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分子分析以及免疫组织化学研究显示，钠、钾和神经递质受体相关通道的概况与持续钠和泄漏电导的重要作用一致。电生理学研究还表明，这些细胞传导机制在节律性呼吸模式的调节中起着关键作用，这是由一组不同的内源性神经化学物质以及生理控制信号（包括二氧化碳和氧气）来调节这些传导。后者的研究的一个特别的重点是阐明呼吸回路活动的神经调节控制的神经元的脑干肾上腺素能系统，这是假设有一个关键的功能，在脑状态依赖性控制呼吸在体内，并与呼吸的病理生理障碍，如那些潜在的婴儿猝死综合征（SIDS）。在体外和原位进行的电生理研究与完整的制备啮齿动物脑干脊髓已经建立了关键的中缝和呼吸回路神经元之间的功能相互作用，并确定了在新生儿和成熟的哺乳动物神经系统中缝神经元的重要调制作用。此外，在体内研究进行了转基因小鼠，缺乏中缝肌钙蛋白能神经元，现在已经表明，呼吸回路功能的异常sertonergic调制导致严重的呼吸不稳定性和高死亡率在出生时，从而建立了sertonergic神经元稳定的体内稳态呼吸的重要作用。脑干呼吸回路的操作，包括多个神经调节控制机制的新模型已制定解释特定的脑干回路组件是如何控制的。我们目前正在采用药理学和光遗传学方法对神经元群体特异性的活动进行操纵，以进一步研究不同网络神经元群体的活动调节如何有助于在不同（病理）生理状态下产生呼吸模式。

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 neuronal synaptic interactions and cellular membrane biophysical properties in the pre-Botzinger complex, including with advanced electrophysiolgical approaches such as the "dynamic clamp", 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. These studies have provided additional evidence that neuronal persistent sodium currents 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 cellular conductance mechanisms are critically involved in the regulation of the rhythmic breathing patterns by a diverse set of endogenous neurochemicals that modulate these conductances as well as by physiological control signals including carbon dioxide and oxygen. A particular focus of these latter studies was elucidating neuromodulatory control of respiratory circuit activity by neurons of the brainstem serotonergic system, which is postulated to have a critical function in brain state-dependent control of breathing in vivo and is associated with pathophysiological disturbances of breathing such as those underlying sudden infant death syndrome (SIDS). Electrophysiological studies performed with intact preparations of the rodent brainstem-spinal cord in vitro and in situ have established critical functional interactions between raphe and respiratory circuit neurons and determined the essential modulatory actions of raphe serotonergic neurons in both the neonatal and mature mammalian nervous systems. Furthermore, in vivo studies were conducted with transgenic mice that lack raphe serotonergic neurons and have now shown that abnormal sertonergic modulation of respiratory circuit function causes severe instabilities of breathing and high mortality at birth, thus establishing the essential role of sertonergic neurons for stable homeostatic breathing in vivo. New models for the operation of brainstem respiratory circuits that incorporate multiple neuromodulatory control mechanisms have been formulated to explain how specific brainstem circuit components are controlled. We are currently employing pharmaco- and opto-genetic approaches for neuron population-specific manipulation of activity to further investigate how regulation of activity of different populations of network neurons contributes to respiratory pattern generation in different (patho)physiological states.