Neural Mechanisms Controlling Breathing In Mammals

控制哺乳动物呼吸的神经机制

• 批准号: 8557015
• 负责人: Jeffrey c Smith
• 金额: $ 115.3万
• 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/8557015
• 关键词: Address; Architecture; Behavior; Brain; Brain Stem; Breathing; Calcium; Carbon Dioxide; Cell Nucleus; Cells; Cellular Membrane; Complex; Disease; Dyes; Elements; Functional Imaging; Generations; Genetic; Goals; Growth; Hybrids; Hypercapnic respiratory failure; Image; In Situ; In Vitro; Kinetics; Label; Laser Scanning Microscopy; Mammals; Mediating; Membrane; Mental Depression; Messenger RNA; Methods; Modeling; Molecular; Molecular Profiling; Motor; Motor Activity; Movement; Neonatal; Nervous system structure; Neurons; Neurotransmitter Receptor; Opioid; Oxygen; Pacemakers; Pattern; Pattern Formation; Physiological; Population; Preparation; Property; Proteins; Protons; Rattus; Regulation; Research; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Serotonin; Signal Transduction; Sleep Apnea Syndromes; Slice; Sodium; Spinal Cord; Sudden infant death syndrome; Synapses; Syndrome; System; Techniques; Testing; Therapeutic; Time; Viral Vector; Work; base; design; excitatory neuron; in vitro activity; in vivo; inhibitory neuron; multi-photon; multidisciplinary; network models; neural circuit; neurochemistry; neurogenesis; neuromechanism; neurophysiology; neuroregulation; novel; operation; optogenetics; raphe nuclei; receptor; reconstruction; relating to nervous system; research study; respiratory; spatiotemporal; transmission process

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, show a profile of leak-type channels consistent with an important general role of these conductances in regulating excitability of respiratory circuit neurons. We have now 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 oscillatory behavior of respiratory circuit neurons. 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 analyzed 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 pharmaco-genetic 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.

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。 实验研究是用新生儿和成熟大鼠分离的原位灌注脑脊髓和体外脑干切片制剂进行的。以前，我们已经确定了包含参与节奏生成的神经元种群的脑干基因座（称为肉毒纤维综合体）。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，以及节奏传播电路中的神经元，利用通过多光子激光扫描显微镜与液体扫描显微镜的功能活性成像同时进行的结构成像，该神经元用荧光型钙化钙敏感蛋白质蛋白和/或/或/或/或/或/或/或/或/或/或/或/或/或/或/或/或/或/或或或或或或或或或或或或或或或或或或或或或或或或或或或一位或/或/或/或/或或或氟化均含量。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，我们已经对神经元活性进行了高分辨率时空成像，并分析了在新生儿啮齿动物前腐蚀前剂中的呼吸神经元的生物物理特性，并在体外进行了节奏传播电路。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，该神经元具有专门的细胞特性，该特性具有产生呼吸振荡的多种机制。对前胞毒剂络合物中神经元突触相互作用和细胞膜生物物理特性的研究，包括使用细胞内记录技术原位和先进的电物质学方法，例如在体外应用的“动态夹具”，继续支持我们的混合型rh and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and toltos in toltos and toltos in n oferntir and nertiage and ofert and of toltir and of the and of toltir and in n of the and nertiage and在 系统。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。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Messenger RNA的RT-PCR分子分析在单个功能上鉴定的神经元中表达，以及免疫组织化学研究，显示了与这些电导在调节呼吸道电路神经元兴奋性兴奋性中的重要一般作用一致的泄漏型通道的特征。现在，我们已经确定了一组专门的瞬态受体电位（TRP）阳离子通道，该通道也可能代表了神经元兴奋性的重要调节剂，而当前的研究则是针对理解这些通道如何有助于呼吸回路神经元的振荡行为的。电生理学研究表明，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来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的调节。这些后者研究的一个特殊重点是阐明了构成脑干raphe核神经元的神经调节控制，构成脑干血清素（5-HT）系统，假定该系统在脑状态依赖性呼吸中具有关键的功能，并且与病理学的疾病造成了这种疾病的疾病依赖性的呼吸症状相关。我们在体外和原位进行的持续电生理研究已经建立了Raphe和呼吸回路神经元之间的关键功能相互作用，并确定了在新生儿和成熟的哺乳动物神经系统中Raphe 5-HT神经元的基本调节作用。以前，我们已经表明，Raphe 5-HT神经元具有缓慢的起搏特性，部分取决于钠和泄漏通道的动力学特性，并且这些起搏性能被证明对于连续调节呼吸网络兴奋性和呼吸节律的产生至关重要。我们现在已经确定，5-HT神经元的活性受二氧化碳/氢离子的调节，以调节体外和原位的呼吸回路活性。我们还分析了如何利用不同种群的呼吸道神经元种群中各种5-HT受体的药理特性，以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，采用新型的药物生成方法应用原位和体内的研究，逆转录核的神经元（RTN）的神经元也具有缓慢的起搏和化学感应性能，也证明了对产生和增生性新型Neural neural neural neural neural neural neural neural neural neural neural neural neural neural neural neural neural neural neural neural Neal neural Neal neural neural Neural neal secornet网络的关键兴奋性调节型的关键兴奋性调节式输入。 因此，已经制定了脑干呼吸回路运行的新模型，这些模型已制定了多种神经调节输入控制机制，以解释特定的脑干电路组件如何受到控制并调节呼吸振荡活动的模式。目前，我们正在采用光遗传学方法来操纵特定神经元种群的活性，以进一步研究不同（PATHO）生理状态的不同网络神经元种群如何促进呼吸模式的产生。