Neural Mechanisms Controlling Breathing In Mammals

针对该项目的主要具体目标的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方法，利用红外和差分干涉对比(IR-DIC)显微镜进行的结构成像与荧光钙敏感染料和/或荧光蛋白标记的神经元的活动模式的功能同步进行。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。用这些方法，我们已经成像并分析了体外培养的新生啮齿动物Pre-Botzinger复合体和节律传递电路中呼吸神经元的活动和生物物理特性，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特性的神经元的兴奋网络，赋予呼吸回路多种产生呼吸振荡的机制。多光子激光扫描显微镜成像的方法目前正在开发中，这种方法可以三维重建前博辛格复合体和其他呼吸网络组件。对前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，包括使用先进的电生理学方法，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形成的，旨在解释完整哺乳动物神经系统中呼吸节奏和模式的产生和控制。这些研究提供了更多的证据，证明神经元持续钠电流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对体外培养的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，发现钠、钾和神经递质受体连接的通道与持续的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，称为任务通道，它是神经元泄漏电导的重要贡献者。电生理学研究还表明，这些细胞电导机制与调节节律呼吸模式的多种内源性神经化学物质以及包括二氧化碳和氧气在内的生理控制信号密切相关。后一项研究的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制控制，该神经元参与化学感觉(二氧化碳相关)调节，以及脑干5-羟色胺能系统的控制，该系统被认为在体内对呼吸的脑状态依赖控制中具有关键功能，并与呼吸的病理生理障碍有关，如婴儿猝死综合征(SIDS)。在体外和原位进行的电生理学研究已经在中缝和呼吸回路神经元之间建立了关键的功能相互作用，并确定了中缝5-羟色胺能神经元在新生和成熟哺乳动物神经系统中的基本调节作用。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持续调节所必需的。在采用新的药物遗传学方法应用于体内和原位的研究中，同样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成部分提供了关键的兴奋性输入，以产生和协调吸气和呼气神经活动。脑干呼吸回路运作的新模型结合了多种神经调节输入控制机制，以解释特定的脑干回路组件是如何被控制和调节呼吸振荡活动的模式的。我们目前正在使用光遗传学方法来操纵特定神经元群体的活动，以进一步研究不同群体的网络神经元如何在不同(病理)生理状态下对呼吸模式的产生做出贡献。

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.