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。本实验采用新生或成年大鼠和小鼠的离体脑干-脊髓原位灌流标本和离体脑干切片标本进行研究。以前，我们已经确定了脑干位点，称为preBotzinger复合体（preBotC），其中包含对呼吸节律产生至关重要的神经元群体。我们已经进一步开发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，利用结构成像与功能活动成像同时进行的多光子激光扫描显微镜标记的荧光遗传编码的钙传感器和/或荧光蛋白的神经元。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膜通道、受体和神经递质相关蛋白表达的分子研究。通过这些方法，我们对神经元活动进行了高分辨率时空成像，并分析了新生啮齿动物preBotC体外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，即节律的产生涉及具有专门细胞特性的神经元兴奋网络，该神经元兴奋网络赋予呼吸回路产生呼吸振荡的神经元电压依赖性机制。通过应用光遗传学方法，我们先前已经确定了具有电压依赖性振荡特性的神经元的关键群体是新生儿和成年啮齿动物神经系统中preBotC中吸气节律产生的底物。在preBotC神经元突触相互作用和细胞膜生物物理特性的研究，包括与细胞内记录技术原位继续支持我们的混合起搏器网络模型，制定从以前的工作，以解释在完整的哺乳动物神经系统的呼吸节律的产生和控制。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兴奋性和抑制性神经元的不同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，我们正在进行的基于光遗传学的转基因小鼠和新型转基因大鼠的研究，涉及抑制性呼吸神经元的光抑制或光激发，已经建立了抑制性微电路的基本作用，包括在呼吸模式生成中的preBotC中。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代表呼吸振荡的产生和控制的关键离子电导机制。我们先前对体外单个功能鉴定神经元中表达的信使RNA进行RT-PCR的分子分析，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）阳离子通道也是神经元兴奋性的重要调节因子，当前的研究旨在了解这些通道如何影响神经元的电生理行为。呼吸回路神经元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）调节这些传导。此外，我们继续进行星形胶质细胞在preBotC中神经回路活动的调节控制中的作用的新研究，包括通过释放信号分子如ATP，其被假设为响应于体内二氧化碳升高（高碳酸血症）或氧气减少（缺氧），包括在病理生理条件下，来激发产生节律的神经元。通过采用选择性干扰神经胶质递质释放或破坏ATP介导的信号传导的病毒载体，我们已经获得了另外的证据，即星形胶质细胞对体内高碳酸血症和缺氧作出反应，以调节preBotC回路的活性，从而稳态地调节呼吸频率，以部分补偿这些生理干扰。在我们以前和正在进行的研究中，采用新的化学发生方法应用于体内，神经元的后斜方核（RTN），具有化学感受特性也被证明提供了一个关键的兴奋性调节输入的核心组成部分的呼吸网络，包括preBotC调节吸气神经活动的产生。我们的新研究表明星形胶质细胞参与preBotC水平的化学感受调节，这使我们提出了新的概念模型，用于关键呼吸回路的生理调节，其中包括多种神经调节控制机制，包括星形胶质细胞机制。我们目前正在扩展我们基于光遗传学的研究，以操纵区域特异性神经元和星形胶质细胞群体的活性，以进一步研究这些不同群体如何在各种（病理）生理状态下促进呼吸神经活性的产生和控制。

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 or mature rats and mice. Previously we have identified the brainstem locus, called the preBotzinger complex (preBotC), that contains populations of neurons critical for respiratory 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 genetically-encoded calcium sensor and/or fluorescent proteins. This imaging approach has facilitated the identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of the expression of neuron membrane 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 preBotC in vitro. These studies are 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 neuronal voltage-dependent mechanisms for producing respiratory oscillations. By applying optogenetic approaches we have previously established that a critical population of glutamatergic neurons with voltage-dependent oscillatory properties is the substrate for inspiratory rhythm generation in the preBotC in the neonatal and adult rodent nervous systems. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the preBotC, including with intracellular recording techniques in situ continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratory rhythm 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, our in-progress optogenetics-based studies with transgenic mice and novel transgenic rats involving photo-inhibition or photo-excitation of inhibitory respiratory neurons have established a fundamental role of inhibitory microcircuits including in the preBotC 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 the generation and control of respiratory oscillations. Our previous 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. In addition, we continue to conduct novel studies of the role of astrocytes in modulatory control of neural circuit activity in the preBotC, including by the release of signaling molecules such as ATP, which is hypothesized to excite the rhythm generating neurons, in response to elevated carbon dioxide (hypercapnia) or reduced oxygen (hypoxia) in vivo, including in pathophysiological conditions. By employing viral-vectors that selectively interfere with the release of glial transmitters or disrupt ATP-mediated signaling, we have obtained additional evidence that astrocytes respond to hypercapnia and hypoxia in vivo to regulate the activity of preBotC circuits to homeostatically adjust the breathing frequency to partially compensate for these physiological disturbances. In our previous and ongoing studies employing novel chemogenetic approaches applied in vivo, neurons of the retrotrapezoid nucleus (RTN) that have chemosensory properties are also demonstrated to provide a critical excitatory modulatory input to core components of the respiratory network including the preBotC to regulate the generation of inspiratory neural activity. Our new studies showing the involvement of astrocytes in chemosensory regulation at the level of the preBotC have led us to propose new conceptual models for the physiological regulation of key respiratory circuits that incorporate multiple neuromodulatory control mechanisms including astrocytic mechanisms. We are currently extending our optogenetics-based studies to manipulate the activity of regionally specific neuronal and astrocyte populations to further investigate how these different populations contribute to the generation and control of respiratory neural activity in various (patho)physiological states.