Neural Mechanisms Controlling Breathing In Mammals

该项目的研究主要集中在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上。实验研究采用新生儿和成年大鼠和小鼠的离体原位灌注脑干-脊髓和离体脑干切片进行。以前，我们已经确定了脑干位点（称为前botzinger复合体）包含参与节奏产生的神经元群。我们进一步开发了这些神经元的实时结构和功能成像方法，以及节律传递回路中的神经元，利用结构成像与功能活动成像同时进行，通过多光子激光扫描显微镜对荧光钙敏感染料和/或荧光蛋白标记的神经元进行成像。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特性的电生理研究，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，我们进行了神经元活动的高分辨率时空成像，并在体外分析了新生啮齿动物pre-Botzinger复合物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，表明节律的产生涉及具有特殊细胞特性的神经元的兴奋性网络，该网络赋予呼吸回路产生呼吸振荡的多种机制。通过应用光遗传学方法，我们已经证实具有电压依赖性振荡特性的谷氨酸能神经元的临界种群是前波青格复合体中吸气节律产生的底物。对pre-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学方法，如体外应用的“动态钳”，继续支持我们的混合起搏器网络模型，该模型是根据之前的工作制定的，用于解释完整哺乳动物神经系统中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以产生呼吸节律和模式，以及测试我们的网络模型的预测。此外，基于光遗传学的研究涉及抑制呼吸神经元的光抑制或光激发，已经确定了抑制微电路在呼吸模式产生中的基本作用，包括前波青格复合物。其他研究提供了额外的证据，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。利用RT-PCR对体外单个功能鉴定神经元中表达的信使RNA进行分子分析，以及我们目前的免疫组织化学和药理学研究，已经确定了一组专门的瞬时受体电位（TRP）阳离子通道，这些通道也代表了神经元兴奋性的重要调节因子，目前的研究旨在了解这些通道如何影响呼吸回路神经元的电生理行为。其他电生理学研究表明，泄漏传导机制在节律性呼吸模式的调节中起着至关重要的作用，通过多种内源性神经化学物质调节这些传导，以及通过包括二氧化碳和氧气在内的生理控制信号。后一项研究的重点是阐明脑干中隔核神经元对呼吸回路活动的神经调节控制，这些神经元构成脑干血清素（5-HT）系统，该系统被认为在体内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（SIDS）。我们在体外和原位进行的持续电生理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，并确定了中缝5-HT神经元在新生儿和成熟哺乳动物神经系统中的基本调节作用。先前我们已经证明，中段5-HT神经元具有缓慢的起搏特性，部分依赖于钠离子和泄漏通道的动力学特性，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。我们还继续分析了不同类型的5-HT受体，特别是5-HT（1a）受体在不同呼吸回路神经元群体上的药理激活如何影响回路活动，以及如何利用这一点来逆转阿片类药物诱导的呼吸抑制，并具有潜在的转化治疗应用。在先前的研究中，采用新的药物遗传学方法在体内和原位应用，后梯形核（RTN）的神经元也具有缓慢的起搏和化学感觉特性，为呼吸网络的核心成分提供关键的兴奋调节输入，以产生和协调吸气和呼气神经活动。因此，结合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，以解释特定脑干回路组件是如何被控制和调节呼吸振荡活动模式的。我们目前正在扩展基于光遗传学的研究，以操纵特定神经元群体的活动，进一步研究不同网络神经元群体如何在各种（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 and mice. 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. By applying optogenetic approaches we have confirmed that a critical population of glutamatergic neurons with voltage-dependent oscillatory properties are the substrate for inspiratory rhythm generation in the pre-Botzinger complex. 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. Furthermore, optogenetics-based studies involving photo-inhibition or photo-excitation of inhibitory respiratory neurons has established a fundamental role of inhibitory microcircuits including in the pre-Botzinger complex in respiratory pattern generation. 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 our current immunohistochemical and pharmacological studies, have identified a specialized set of transient receptor potential (TRP) cationic channels that 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 also continued to analyze how pharmacological activation of various types of 5-HT receptors, especially 5-HT(1a) receptors, on different populations of respiratory circuit neurons affects circuit activity and how this 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 extending the optogenetics-based studies to manipulate activity of specific neuronal populations to further investigate how different populations of network neurons contribute to respiratory pattern generation in various (patho)physiological states.