Neural Mechanisms Controlling Breathing In Mammals

控制哺乳动物呼吸的神经机制

• 批准号: 8342214
• 负责人: Jeffrey c Smith
• 金额: $ 117.06万
• 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/8342214
• 关键词: Address; Architecture; Brain; Brain Stem; Breathing; Calcium; Carbon Dioxide; Cell Nucleus; Cells; Cellular Membrane; Complex; Development; Disease; Dyes; Elements; Fluorescence; Functional Imaging; Generations; Genetic; Goals; Growth; Hybrids; Hypercapnic respiratory failure; Image; In Situ; In Vitro; Kinetics; Label; Laser Scanning Microscopy; Link; Mammals; Mediating; Membrane; Messenger RNA; Methods; Modeling; Molecular; Molecular Profiling; Motor; Motor Activity; Movement; Neonatal; Nervous system structure; Neurons; Neurotransmitter Receptor; Nomarski Interference Contrast Microscopy; Oxygen; Pacemakers; Pattern; Pattern Formation; Physiological; Population; Potassium; Potassium Channel; Preparation; Property; Proteins; Rattus; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Serotonin; Signal Transduction; Sleep Apnea Syndromes; Slice; Sodium; Sodium Channel; Spinal Cord; Sudden infant death syndrome; Synapses; Syndrome; System; Time; Viral Vector; Work; base; design; excitatory neuron; imaging modality; in vivo; multi-photon; multidisciplinary; network models; neural circuit; neurochemistry; neurogenesis; neuromechanism; neurophysiology; neuroregulation; novel; operation; reconstruction; relating to nervous system; research study; respiratory; 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 by infrared and differential interference contrast (IR-DIC) microscopy performed simultaneously with functional of activity patterns of the neurons labeled with fluorescence 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 imaged the activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro, 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 multiple mechanisms for producing respiratory oscillations. Methods for imaging by multi-photon laser scanning microscopy that allow three-dimensional reconstruction of the pre-Botzinger complex and other respiratory network components are currently under development. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-Botzinger complex, including with advanced electrophysiolgical approaches such as the "dynamic clamp", continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratoy rhythm and pattern in the intact mammalian nervous system. These studies have provided additional evidence that neuronal persistent sodium currents and potassium leak 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 sodium, potassium, and neurotransmitter receptor-linked channels consistent with an important role of persistent sodium and potassium leak conductances. We have now identified a specialized class of two-pore domain potassium channels, called TASK channels, that are important contributors to neuronal leak conductance. Electrophysiological studies have also demonstrated that these cellular 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 retrotrapezoid nucleus (RTN), which is critically involved in chemosensory (carbon dioxide-related) regulation, and control by the brainstem serotonergic 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 those underlying sudden infant death syndrome (SIDS). 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 serotonergic neurons in both the neonatal and mature mammalian nervous systems. Raphe neurons were shown to have slow pacemaking properties dependent in part on the kinetic properties of sodium channels, and these pacemaking properties were demonstrated to be essential for continuous modulation of respiratory network excitability and respiratory rhythm generation. In studies employing novel pharmaco-genetic approaches applied in situ and in vivo, RTN neurons that also have slow pacemaking properties were shown to provide a critical excitatory input to core components of the respiratory network for generation and coordination of inspiratory and expiratory neural activity. 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，以及节奏传播循环中的神经元，利用红外和差分干扰对比（IR-DIC）显微镜同时进行的结构成像（IR-DIC）显微镜同时执行的神经元功能与液体含量的神经元的功能同时进行。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，我们已经成像了活性并分析了新生儿啮齿动物神经元的生物物理特性，并在体外进行了botzinger Predzinger复合物和节奏传播回路，迄今为止，迄今为止，节奏的最直接的实验证据提供了具有专用细胞的兴奋性网络，这些兴奋性网络涉及具有多种式培养基的神经元素的兴奋性网络。通过多光子激光扫描显微镜进行成像的方法，该方法允许对肉毒前复合物和其他呼吸网络组件进行三维重建。对前体综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，包括采用先进的电物质学方法，例如“动态夹具”，继续支持我们的混合起搏器网络模型，该模型是从先前的工作中提出的，以解释rhythm和Pattern in Pertintay Rhythm和Pattern in Pottact of the Intact cottact cottact Mantact Mymal Mymal Mamamal Mammal Mammal Mammal Mammal Mammal Mammal Mammal Mammal Mammal Mammal systry。这些研究提供了其他证据，表明神经元持续的钠电流和钾泄漏电导代表了生成和控制呼吸振荡的关键离子电导机制。用在体外功能鉴定的神经元中表达的Messenger RNA的RT-PCR以及免疫组织化学研究表明，钠，钾和神经递质受体链接的通道的特征是一致的，与持久性钠和钾漏液的重要作用一致。现在，我们已经确定了一类专业的两孔域钾通道，称为任务通道，这是神经元泄漏电导的重要贡献者。电生理研究还表明，这些细胞电导机制与一组多种内源性神经化学物质对节奏呼吸模式进行了严格的调节，这些神经化学物质通过调节这些电导以及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。这些后一项研究的一个特殊重点是阐明了通过脑干逆转录核（RTN）神经元对呼吸道回路活性的神经调节控制，该核（RTN）与化学水敏（二氧化碳相关）调节（二氧化碳相关）调节，以及由脑词干系统的态度与脑部的态度相关，在脑形角质系统中与脑部的态度相关，并与脑部的相关性相关，该脑部与脑部的相关性相关，该脑部与脑部的相关性相关方面的相关功能与脑部的相关性相关，从而与之控制。呼吸的病理生理障碍，例如那些猝死综合征（SIDS）的呼吸。在体外和原位进行的电生理研究已经在Raphe和呼吸道神经元之间建立了关键的功能相互作用，并确定了在新生儿和成熟的哺乳动物神经系统中Raphe 5-羟色胺能神经元的基本调节作用。 Raphe神经元显示出缓慢的起搏特性，部分取决于钠通道的动力学特性，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奏产生至关重要。在采用原位和体内应用的新型药物遗传学方法的研究中，也证明其也具有缓慢的起搏性能的RTN神经元可为呼吸网络的核心成分提供关键的兴奋性输入，以生成和协调灵感和呼气性神经活动。 已经制定了包含多种神经调节输入控制机制的脑干呼吸回电运行的新模型，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。