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，利用多光子激光扫描显微镜对标记有荧光钙敏感染料和/或荧光蛋白的神经元进行结构成像和功能活动成像。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。利用这些方法，我们对神经元活动进行了高分辨率的时空成像，并在体外分析了新生啮齿动物Pre-Botzinger复合体和节律传递回路中呼吸神经元的生物物理特性。这些研究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，该网络赋予呼吸回路多种产生呼吸振荡的机制。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，包括细胞内原位记录技术和先进的电生理方法，如体外应用的“动态钳”，继续支持我们从以前的工作中形成的混合起搏器-网络模型，该模型解释了完整哺乳动物神经系统呼吸节律和模式的产生和控制。基于原位应用的细胞内记录方法正在进行的研究正在详细分析不同的兴奋性和抑制性神经元群体如何相互作用来产生呼吸节律和模式，以及测试我们的网络模型的预测。其他研究提供了更多证据，表明神经元持续钠电流和几种类型的泄漏或背景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神经元表达的信使RNA进行分子图谱分析，以及免疫组织化学研究，已经确定了一组特殊的瞬时受体电位(TRP)阳离子通道，这些通道也可能是神经元兴奋性的重要调节因素，目前的研究旨在了解这些通道如何对呼吸回路神经元的电生理行为做出贡献。其他电生理学研究表明，泄漏电导机制在调节有节奏的呼吸模式中起关键作用，参与调节这些电导的各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。后者的重点是阐明脑干中缝核神经元对呼吸回路活动的神经调制控制，该核团构成脑干5-羟色胺(5-HT)系统，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，如潜在的婴儿猝死综合征(SDS)。我们在体外和原位进行的持续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-羟色胺神经元在新生和成熟哺乳动物神经系统中的基本调制作用。以前我们已经证明，中缝5-羟色胺神经元具有缓慢的起搏特性，部分依赖于钠通道和泄漏通道的动力学特性，这些起搏特性被证明是持续调节呼吸网络兴奋性和呼吸节律产生所必需的。我们现在已经证实，在体外和原位，5-羟色胺神经元的活动受到二氧化碳/氢离子的调节，以实现呼吸回路活动的动态平衡调节。我们还继续分析如何利用不同呼吸回路神经元上各种类型的5-羟色胺受体的药理学特性来逆转阿片类药物引起的呼吸抑制，并具有潜在的翻译治疗应用。在利用新的药物遗传学方法应用于体内和原位的研究中，同样具有缓慢起搏和化学感觉特性的斜方后核(RTN)神经元也被证明为呼吸网络的核心成分提供关键的兴奋性调制输入，以产生和协调吸气和呼气神经活动。相应地，结合了多种神经调节输入控制机制的脑干呼吸回路运行的新模型已经形成，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 performed simultaneously with functional activity imaging by multi-photon laser scanning microscopy of the neurons labeled with fluorescent 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 performed high-resolution spatiotemporal imaging of neuron activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro. These studies have provided 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. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-Botzinger complex, including with intracellular recording techniques in situ and advanced electrophysiolgical approaches such as the "dynamic clamp" applied in vitro, continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratory rhythm and pattern in the intact mammalian nervous system. Studies in progress based on intracellular recording approaches applied in situ are analyzing in detail how distinct populations of excitatory and inhibitory neurons interact to generate the respiratory rhythm and pattern as well as to test predictions of our network models. Other studies have provided additional evidence that neuronal persistent sodium currents and several types of leak or background 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, have identified a specialized set of transient receptor potential (TRP) cationic channels that may also represent important regulators of neuron excitability and current studies are directed toward understanding how these channels may contribute to electrophysiological behavior of respiratory circuit neurons. Other electrophysiological studies have demonstrated that leak 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 raphe nucleus that constitute the brainstem serotonin (5-HT) 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 thosse underlying sudden infant death syndrome (SIDS). Our continuing 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 5-HT neurons in both the neonatal and mature mammalian nervous systems. Previously we have shown that raphe 5-HT neurons have slow pacemaking properties dependent in part on the kinetic properties of sodium and leak channels, and these pacemaking properties were demonstrated to be essential for continuous modulation of respiratory network excitability and respiratory rhythm generation. We have now established that the activity of 5-HT neurons is regulated by carbon dioxide/hydrogen ion for homeostatic regulation of respiratory circuit activity in vitro and in situ. We have also continued to analyze how the pharmacological properties of various types of 5-HT receptors on different populations of respiratory circuit neurons can be exploited to reverse opioid-induced depression of breathing with potential translational therapeutic applications. In previous studies employing novel pharmacogenetic approaches applied in situ and in vivo, neurons of the retrotrapezoid nucleus (RTN) that also have slow pacemaking and chemosensory properties were also shown to provide a critical excitatory modulatory input to core components of the respiratory network for generation and coordination of inspiratory and expiratory neural activity. Accordingly 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.