Neural Mechanisms Controlling Breathing In Mammals

控制哺乳动物呼吸的神经机制

• 批准号: 7969555
• 负责人: Jeffrey c Smith
• 金额: $ 111.76万
• 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/7969555
• 关键词: Architecture; Arts; Birth; Brain; Brain Stem; Breathing; Calcium; Carbon Dioxide; Cellular Membrane; Complex; Development; Dyes; Functional Imaging; Generations; Genetic; Goals; Growth; Hybrids; Image; In Situ; In Vitro; Label; Link; Mammals; Messenger RNA; Methods; Modeling; Molecular; Molecular Profiling; Movement; Neonatal; Nervous system structure; Neurons; Neurotransmitter Receptor; Operative Surgical Procedures; Oxygen; Pacemakers; Pattern; Physiological; Population; Potassium; Preparation; Property; Rattus; Regulation; Research; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Signal Transduction; Site; Slice; Sodium; Spinal Cord; Sudden infant death syndrome; Synapses; Synaptic Transmission; System; Time; Transgenic Mice; Work; design; fluorescence imaging; in vivo; mortality; multi-photon; multidisciplinary; network models; neural circuit; neurochemistry; neurogenesis; neuromechanism; neurophysiology; neuroregulation; novel; receptor expression; reconstruction; relating to nervous system; research study; respiratory; transmission process

Research 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 developed novel methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing infrared and differential interference contrast (IR-DIC) imaging performed simultaneously with fluorescence imaging of activity patterns of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of neuron channel and receptor expression. 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 a network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. Methods for multi-photon imaging that will allow three-dimensional reconstruction of this network in the pre-Botzinger complex 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, 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 leak conductances. Electrophysiological studies have also demonstrated that these cellular conductance mechanisms are critically involved in the regulation of the 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 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 with intact preparations of the rodent brainstem-spinal cord in vitro and in situ have established critical functional interactions between raphe and respiratory circuit neurons and determined the essential modulatory actions of raphe serotonergic neurons in both the neonatal and mature mammalian nervous systems. Furthermore, in vivo studies were conducted with transgenic mice that lack raphe serotonergic neurons and have now shown that abnormal sertonergic modulation of respiratory circuit function causes severe instabilities of breathing and high mortality at birth, thus establishing the essential role of sertonergic neurons for stable homeostatic breathing in vivo. New models for the operation of brainstem respiratory circuits that incorporate multiple neuromodulatory control mechanisms have been formulated to explain how specific brainstem circuit components are controlled. We are currently employing pharmaco- and opto-genetic approaches for neuron population-specific manipulation of activity to further investigate how regulation of activity of different populations of network neurons contributes to respiratory pattern generation in different (patho)physiological states.

研究重点是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）。我们进一步开发了这些神经元的实时结构和功能成像的新方法，以及节奏传播电路中的神经元，利用红外和差异干扰对比度（IR-DIC）成像（IR-DIC）成像（IR-DIC）与钙敏感染料的神经元的荧光成像同时进行。这种成像方法促进了对生物物理和突触特性的电生理研究以及神经元通道和受体表达的分子研究的识别。通过这些方法，我们已经成像了新生儿啮齿动物前植物前复合物和体外节奏传播循环的呼吸神经元的活性和分析的生物物理特性，从而提供了迄今为止节奏的最直接的实验证据，该证据提供了具有多种呼吸道循环系统的专用细胞性能的神经元网络，以产生多种呼吸道循环。当前正在开发中，可以在前博客综合体中进行三维重建的多光子成像方法。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。用在单个功能鉴定的神经元中表达的使者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与持久钠和泄漏电导的重要作用一致。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以及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脑干血清素能系统神经元对呼吸道回路活性的神经调节性控制，该神经元被认为在脑状态依赖体内呼吸中具有关键功能，并且与呼吸的病理生理学障碍有关，例如那些潜在的突然婴儿死亡综合征（s）。在体外和原位啮齿动物​​脑脊髓的完整制备进行的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，并确定了新生儿和成熟的哺乳动物神经系统中Raphe血清素能神经元的必要调节作用。此外，对缺乏Raphe血清素能神经元的转基因小鼠进行了体内研究，现在已经表明，呼吸回路功能的异常静脉局能调节导致出生时呼吸和高死亡率的严重不稳定性，从而确立了静态神经元在Vivivo中的稳定稳定性抑制作用的重要作用。已经制定了包含多种神经调节控制机制的脑干呼吸回电运行的新模型，以解释如何控制特定的脑干电路组件。目前，我们正在采用药物和光遗传学方法来对活性进行神经元人群特异性操纵，以进一步研究不同网络神经元不同人群活动的调节如何有助于不同（PATHO）生理状态的呼吸模式产生。