Neural Mechanisms Controlling Breathing In Mammals

针对该项目主要具体目标的研究侧重于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。我们已经进一步利用了这些神经元的实时结构和功能成像的方法，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）显微镜结构成像与功能的活动模式的神经元标记的荧光钙敏感染料和/或荧光蛋白同时进行。这种成像方法促进了呼吸回路神经元的识别，用于生物物理和突触特性的电生理学研究以及神经元通道、受体和神经递质相关蛋白表达的分子研究。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的啮齿动物前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兴奋性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。目前正在开发通过多光子激光扫描显微镜进行成像的方法，该方法允许对前Botzinger复合体和其他呼吸网络组件进行三维重建。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。在体外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性钠和钾泄漏电导的重要作用一致。我们现在已经确定了一类特殊的双孔结构域钾通道，称为ASK通道，是神经元漏导的重要贡献者。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。后者研究的一个特别重点是阐明脑干后斜方核（RTN）神经元对呼吸回路活动的神经调节控制，RTN神经元在化学感觉中起关键作用。（二氧化碳相关的）调节和脑干神经元能系统的控制，它被认为在大脑状态中具有关键功能-依赖于体内呼吸的控制，并与呼吸的病理生理学紊乱有关，如婴儿猝死综合征（SIDS）的基础。在体外和原位进行的电生理学研究已经建立了中缝和呼吸回路神经元之间的关键功能相互作用，并确定了在新生儿和成熟哺乳动物神经系统中缝神经元能神经元的基本调节作用。中缝神经元具有缓慢的起搏特性，部分依赖于钠通道的动力学特性，这些起搏特性被证明是呼吸网络兴奋性和呼吸节律产生的连续调节所必需的。在采用原位和体内应用的新型药物遗传学方法的研究中，也具有缓慢起搏特性的RTN神经元被证明为呼吸网络的核心组件提供关键的兴奋性输入，以产生和协调吸气和呼气神经活动。 脑干呼吸回路的操作，包括多个神经调节输入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和调节模式的呼吸振荡活动。我们目前正在采用光遗传学方法来操纵特定神经元群体的活性，以进一步研究不同群体的网络神经元如何在各种（病理）生理状态下促进呼吸模式的产生。

Research addressing the main specific aims of this project 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 exploited methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing structural imaging by infrared and differential interference contrast (IR-DIC) microscopy performed simultaneously with functional of activity patterns of the neurons labeled with fluorescence calcium-sensitive dyes and/or fluorescent proteins. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of expression of neuron channels, receptors, and neurotransmitter-related proteins. 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 an excitatory network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. Methods for imaging by multi-photon laser scanning microscopy that allow three-dimensional reconstruction of the pre-Botzinger complex and other respiratory network components 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 in vitro, 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 potassium leak conductances. We have now identified a specialized class of two-pore domain potassium channels, called TASK channels, that are important contributors to neuronal leak conductance. Electrophysiological studies have also demonstrated that these cellular conductance mechanisms are critically involved in the regulation of 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 retrotrapezoid nucleus (RTN), which is critically involved in chemosensory (carbon dioxide-related) regulation, and control by 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 in vitro and in situ have established critical functional interactions between raphe and respiratory circuit neurons and have determined the essential modulatory actions of raphe serotonergic neurons in both the neonatal and mature mammalian nervous systems. Raphe neurons were shown to have slow pacemaking properties dependent in part on the kinetic properties of sodium channels, and these pacemaking properties were demonstrated to be essential for continuous modulation of respiratory network excitability and respiratory rhythm generation. In studies employing novel pharmaco-genetic approaches applied in situ and in vivo, RTN neurons that also have slow pacemaking properties were shown to provide a critical excitatory input to core components of the respiratory network for generation and coordination of inspiratory and expiratory neural activity. New models for the operation of brainstem respiratory circuits that incorporate multiple neuromodulatory input control mechanisms have been formulated to explain how specific brainstem circuit components are controlled and regulate patterns of respiratory oscillatory activity. We are currently employing optogenetic approaches for manipulation of activity of specific neuronal populations to further investigate how different populations of network neurons contribute to respiratory pattern generation in various (patho)physiological states.