Neural Mechanisms Controlling Breathing In Mammals

控制哺乳动物呼吸的神经机制

• 批准号: 9563104
• 负责人: Jeffrey c Smith
• 金额: $ 166.63万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9563104
• 关键词: Address; Adult; Architecture; Astrocytes; Behavior; Biophysics; Brain; Brain Stem; Breathing; Calcium; Carbon Dioxide; Cations; Cell Nucleus; Cells; Cellular Membrane; Closure by clamp; Electrophysiology (science); Elements; Frequencies; Functional Imaging; Generations; Glutamates; Goals; Hybrids; Hypercapnia; Hypercapnic respiratory failure; Hypoxia; Image; In Situ; In Vitro; Ion Channel; Label; Laser Scanning Microscopy; Mammals; Mediating; Membrane; Messenger RNA; Methods; Molecular; Molecular Profiling; Motor; Motor Activity; Movement; Mus; Neonatal; Nervous system structure; Neuraxis; Neurons; Neurotransmitters; Oxygen; Pacemakers; Pattern; Pattern Formation; Periodicity; Pharmacogenetics; Pharmacology Study; Physiological; Population; Preparation; Property; Proteins; Rattus; Regulation; Research; Resolution; Respiration Disorders; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Signal Transduction; Signaling Molecule; Sleep Apnea Syndromes; Slice; Sodium; Spinal Cord; Sudden infant death syndrome; Synapses; Syndrome; System; Techniques; Testing; Time; Transgenic Mice; Transgenic Organisms; Viral Vector; Work; base; biophysical analysis; biophysical properties; design; excitatory neuron; experimental study; imaging approach; imaging study; in vivo; inhibitory neuron; multidisciplinary; multiphoton imaging; network models; neural circuit; neurochemistry; neuromechanism; neurophysiology; neuroregulation; novel; operation; optogenetics; physiologic model; preBotzinger complex; receptor; reconstruction; relating to nervous system; respiratory; response; sensor; spatiotemporal; transmission process; voltage

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.

研究该项目的主要特定目的的研究集中在啮齿动物脑干中产生呼吸节奏和神经活动模式的细胞和电路机制。实验研究是用新生大鼠或成熟大鼠和小鼠的原位原位灌注脑脊髓和体外脑干切片制剂进行的。以前，我们已经确定了脑干基因座，称为前毒剂络合物（BOTC），其中包含对呼吸节奏产生至关重要的神经元的种群。我们采用了这些神经元的实时结构和功能成像的进一步利用方法，以及节奏传播循环中的神经元，利用由多光子激光扫描的功能活性成像同时执行的结构成像，该功能活性成像与荧光属性元素传感器的神经元标记的神经元标记的神经元和/或/calmented carcented carcented calcented carcented carcented calcented和/calmented calmented carcented and/calment calment calment calments scan。这种成像方法促进了呼吸回路神经元的鉴定，用于生物物理和突触特性的电生理研究，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，我们对神经元活性进行了高分辨率时空成像，并分析了新生儿啮齿动物的体外前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，即节奏的产生涉及具有专门细胞特性的神经元的兴奋性网络，这些神经元具有专门的细胞特性，该特性赋予了具有神经元电压依赖性机制，用于产生呼吸振荡。通过采用光遗传学方法，我们确定具有依赖电压依赖性振荡特性的谷氨酸能神经元的关键群体是新生儿和成人啮齿动物神经系统中BOTC中灵感节奏产生的底物。对BOTC中神经元突触相互作用和细胞膜生物物理特性的研究，包括在体外应用的原位和先进的电生理学方法（例如“动态夹具”），继续支持我们的Hybrid Pacemaker-Network模型，从而使我们的混合起搏器NETWork模型继续进行了以前的工作，该模型从先前的工作中表现出了rhym rhy rhy rhy ry ry ry ry ny rym ins nerm rhy rym ins ofermant offro in consed and in n hysbrid network模型继续使用。基于原位应用的细胞内记录方法进行的研究详细分析了兴奋性和抑制性神经元的不同种群如何相互作用以产生呼吸节奏和模式以及测试网络模型的预测。此外，我们针对转基因小鼠和新的转基因大鼠的新基于光遗传学的研究，涉及抑制照片或抑制性呼吸神经元的照相，已经确立了抑制性微电路的基本作用，包括在呼吸模式生成中，包括前BOTC中的抑制性微电路。其他研究还提供了其他证据，表明神经元持续的钠电流和几种类型的泄漏或背景电导代表了生成和控制呼吸振荡的关键离子电导机制。用在单个功能上鉴定的神经元以及我们当前的免疫组织化学和药理学研究中表达的Messenger RNA的RT-PCR分子分析已经确定了一组专业的瞬态受体电位（TRP）阳离子通道（TRP）阳离子通道，这些通道也可以对这些通道的神经兴奋性和当前的神经性研究对这些通道的贡献贡献对电动性的行为贡献，从而可以对这些通道的行为进行贡献。其他电生理研究表明，泄漏电导机制与一组多种内源性神经化学物质以及通过包括二氧化碳和氧气在内的生理控制信号调节这些电导以及生理控制信号的多种内源性神经化学物质对节奏呼吸模式进行了严格的参与。此外，我们对星形胶质细胞在pre-botC中神经回路活性中的作用进行了新的研究，包括通过释放诸如ATP的信号分子（如ATP）（假设氧化氧化物（高碳酸酯）或氧化氧化物升高）（VIV氧化物），从而激发节奏产生神经元的节律神经元。 我们通过采用病毒向量来选择性地干扰神经胶质发射机的释放或破坏ATP介导的信号传导，使星形胶质细胞对体内的高碳酸盐和低氧反应反应，以调节BOTC电路前的活性，以调节稳态的呼吸频率，从而对呼吸频率进行部分补偿，从而对这些生理学扰动进行部分补偿。 在我们先前采用原位和体内应用的新型药物遗传学方法的研究中，还证明具有化学感应性能的逆转肌核（RTN）的神经元还显示出对呼吸网络的核心成分提供关键的兴奋性调节性输入，包括potc的呼吸网络，包括per potc，以调节灵感化神经活动的产生。 我们的新研究表明，星形胶质细胞参与了BOTC级别的化学感觉调节，这使我们提出了新的概念模型，以对关键呼吸回路的生理调节，这些模型结合了包括星形胶质细胞机制在内的多种神经调节控制机制。目前，我们正在扩展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种群的活性，以进一步研究这些不同人群如何在各种（PATHO）生理状态下产生和控制呼吸神经活动。