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，称为前Botzinger复合体（前BotC），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。这种成像方法有利于识别呼吸回路神经元的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的表达的分子研究。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，并分析了新生啮齿类动物体外前BotC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，即节律的产生涉及具有专门细胞特性的神经元的兴奋性网络，其赋予呼吸回路用于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前BotC中吸气节律产生的底物。前BotC的神经元突触相互作用和细胞膜生物物理特性的研究，包括细胞内记录技术原位和先进的电生理方法，如“动态钳”在体外应用，继续支持我们的混合起搏器网络模型，制定从以前的工作，以解释完整的哺乳动物神经系统中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，我们对涉及抑制性呼吸神经元的光抑制或光激发的转基因小鼠和新型转基因大鼠进行的新的基于光遗传学的研究已经确立了抑制性微电路的基本作用，包括在呼吸模式产生中的前BotC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了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的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）阳离子通道也是神经元兴奋性的重要调节剂，目前的研究旨在了解这些通道如何有助于呼吸回路的电生理行为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，我们还对星形胶质细胞在前BotC中神经回路活动的调节控制中的作用进行了新的研究，包括释放信号分子如ATP，这被假设为刺激节律产生神经元，以响应体内二氧化碳升高（高碳酸血症）或氧气减少（缺氧）。 我们已经确定，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，星形胶质细胞在体内对高碳酸血症和缺氧作出反应，以调节前BotC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。 在我们以前的研究中，采用新的药物遗传学方法应用于原位和体内，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，包括前BotC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前BotC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的群体如何在各种（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 pre-Botzinger complex (pre-BotC), 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 identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of expression of neuron membrane channels, receptors, and neurotransmitter-related proteins. With these approaches, we have performed high-resolution spatio-temporal imaging of neuron activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-BotC 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 neuronal voltage-dependent mechanisms for producing respiratory oscillations. By applying optogenetic approaches we have established that a critical population of glutamatergic neurons with voltage-dependent oscillatory properties is the substrate for inspiratory rhythm generation in the pre-BotC in the neonatal and adult rodent nervous system. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-BotC, 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 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 new 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 pre-BotC 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. In addition, we have conducted novel studies of the role of astrocytes in modulatory control of neural circuit activity in the pre-BotC, 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. We have determined by employing viral-vectors that selectively interfere with release of glial transmitters or disrupt ATP-mediated signaling that astrocytes respond to hypercapnia and hypoxia in vivo to regulate the activity of pre-BotC circuits to homeostatically adjust the breathing frequency to partially compensate for these physiological disturbances. In our previous studies employing novel pharmacogenetic approaches applied in situ and in vivo, neurons of the retrotrapezoid nucleus (RTN) that have chemosensory properties were also shown to provide a critical excitatory modulatory input to core components of the respiratory network including the pre-BotC to regulate generation of inspiratory neural activity. Our new studies showing involvement of astrocytes in chemosensory regulation at the level of the pre-BotC 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 activity of regionally specific neuronal and astrocyte populations to further investigate how these different populations contribute to generation and control of respiratory neural activity in various (patho)physiological states.