Neural Mechanisms Controlling Breathing In Mammals

研究集中在细胞和电路机制产生呼吸节律和神经活动模式的脑干啮齿类动物。实验研究采用新生大鼠和成熟大鼠的离体原位灌注脑干-脊髓和离体脑干切片进行。以前，我们已经确定了脑干位点（称为前botzinger复合体）包含参与节奏产生的神经元群。我们进一步开发了这些神经元以及节奏传输电路中的神经元的实时结构和功能成像的新方法，利用红外和微分干涉对比（IR-DIC）成像与钙敏感染料标记的神经元活动模式的荧光成像同时进行。这种成像方法有助于识别呼吸回路神经元，用于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。通过这些方法，我们对新生啮齿动物的前波青格复合物和体外节律传递回路中呼吸神经元的活性进行了成像和生物物理特性分析，提供了迄今为止最直接的实验证据，证明节律产生涉及具有特殊细胞特性的神经元网络，这些神经元网络赋予呼吸回路产生呼吸振荡的多种机制。目前正在开发的多光子成像方法将允许在波青格前复合体中对该网络进行三维重建。对前波青格复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，包括先进的电生理学方法，如“动态钳”，继续支持我们的混合起搏器网络模型，该模型是根据之前的工作制定的，用于解释完整哺乳动物神经系统中呼吸节奏和模式的产生和控制。这些研究提供了额外的证据，证明神经元持续钠电流和钾泄漏电导是产生和控制呼吸振荡的关键离子电导机制。在单个功能鉴定的神经元中表达的信使RNA的RT-PCR分子分析，以及免疫组织化学研究，显示了钠、钾和神经递质受体连接通道的特征，与持续钠和泄漏传导的重要作用一致。电生理学研究也表明，这些细胞电导机制在节律性呼吸模式的调节中起着至关重要的作用，通过多种内源性神经化学物质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。这些后一项研究的重点是阐明脑干5 -羟色胺能系统神经元对呼吸回路活动的神经调节控制，该系统被认为在体内呼吸的脑状态依赖控制中具有关键功能，并与呼吸的病理生理障碍有关，如潜在的婴儿猝死综合征（SIDS）。在体外和原位对啮齿类动物脑干-脊髓的完整制备进行的电生理学研究已经确立了中缝和呼吸回路神经元之间的关键功能相互作用，并确定了中缝5 -羟色胺能神经元在新生儿和成熟哺乳动物神经系统中的基本调节作用。此外，对缺乏5 -羟色胺能神经元的转基因小鼠进行的体内研究表明，呼吸回路功能的异常5 -羟色胺能调节导致严重的呼吸不稳定和出生时的高死亡率，从而确定了5 -羟色胺能神经元在体内稳定的稳态呼吸中的重要作用。脑干呼吸回路运作的新模型包含了多种神经调节控制机制，用以解释特定脑干回路组件是如何被控制的。我们目前正在采用药物和光遗传学方法对神经元群体的活动进行特异性操作，以进一步研究不同网络神经元群体的活动调节如何在不同（病理）生理状态下促进呼吸模式的产生。

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.