Neural Mechanisms Controlling Breathing In Mammals

该项目的研究主要集中在啮齿动物脑干中产生呼吸节律和神经活动模式的细胞和电路机制上。实验研究采用新生或成熟大鼠和小鼠的离体原位灌注脑干-脊髓和离体脑干切片进行。先前我们已经确定了脑干位点，称为前波青格复合体（pre-Botzinger complex，简称pre-BotC），其中包含对呼吸节律产生至关重要的神经元群。我们进一步开发了这些神经元的实时结构和功能成像方法，以及节律传递电路中的神经元，利用结构成像与功能活动成像同时进行，通过多光子激光扫描显微镜对带有荧光遗传编码钙传感器和/或荧光蛋白标记的神经元进行成像。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，以及神经元膜通道、受体和神经递质相关蛋白表达的分子研究。利用这些方法，我们对体外培养的新生啮齿动物进行了神经元活动的高分辨率时空成像，并分析了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，表明节律的产生涉及具有特殊细胞特性的神经元的兴奋性网络，使呼吸回路具有产生呼吸振荡的神经元电压依赖机制。通过应用光遗传学方法，我们已经确定了具有电压依赖性振荡特性的谷氨酸能神经元的关键种群是新生和成年啮齿动物神经系统中botc前吸气节律产生的基础。神经突触相互作用和细胞膜生物物理特性的研究，包括细胞内原位记录技术和先进的电生理学方法，如体外应用的“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律的产生和控制。基于原位应用的细胞内记录方法的研究正在详细分析不同的兴奋性和抑制性神经元群体如何相互作用以产生呼吸节律和模式，以及测试我们的网络模型的预测。此外，我们新的基于光遗传学的转基因小鼠和新型转基因大鼠的研究涉及光抑制或光激发的抑制性呼吸神经元，已经确定了抑制性微电路在呼吸模式生成中的基本作用，包括前botc。其他研究提供了额外的证据，表明神经元持续钠电流和几种类型的泄漏或背景电导是产生和控制呼吸振荡的关键离子电导机制。利用RT-PCR对体外单个功能鉴定神经元中表达的信使RNA进行分子分析，以及我们目前的免疫组织化学和药理学研究，已经确定了一组专门的瞬时受体电位（TRP）阳离子通道，这些通道也代表了神经元兴奋性的重要调节因子，目前的研究旨在了解这些通道如何影响呼吸回路神经元的电生理行为。其他电生理学研究表明，泄漏传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传导，以及通过包括二氧化碳和氧气在内的生理控制信号。此外，我们对星形胶质细胞在botc前神经回路活动的调节控制中的作用进行了新的研究，包括释放信号分子，如ATP，它被假设为激活产生节律的神经元，以响应体内二氧化碳升高（高碳酸血症）或氧气减少（缺氧）。我们利用病毒载体选择性地干扰胶质递质的释放或破坏atp介导的信号，确定了星形胶质细胞对体内高碳酸血症和缺氧的反应，以调节botc前回路的活性，以自我平衡地调节呼吸频率，以部分补偿这些生理干扰。在我们之前的研究中，采用了新的药物遗传学方法在原位和体内应用，具有化学感觉特性的后梯形核（RTN）神经元也被证明为呼吸网络的核心成分提供关键的兴奋性调节输入，包括前botc，以调节吸气神经活动的产生。我们的新研究表明星形胶质细胞参与了前botc水平的化学感觉调节，这使我们提出了关键呼吸回路生理调节的新概念模型，该模型包含多种神经调节控制机制，包括星形胶质细胞机制。我们目前正在扩展我们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群的活动，以进一步研究这些不同的群体如何在各种（病理）生理状态下促进呼吸神经活动的产生和控制。

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.