Carotid afferent and parafacial neuronal excitatory effects on breathing

描述(由申请人提供)： 呼吸网络中的神经元调节和协调呼吸泵和呼吸道肌肉的激活。因此，肺通气量是由这些神经元的兴奋性和抑制性输入的平衡决定的。长期以来，人们一直认为呼吸的主要兴奋性动力来自于颅内的CO2-H化学感受器。然而，颈动脉体失神经(CBD)后严重的低通气量和中枢CO2-H化疗敏感性的降低表明，颈动脉化学感受器传入提供了主要的兴奋性驱动。尽管存在完整和高度敏感的颅内化学感受器，但CBD如何导致低通气量，以及CBD后两周或更长时间的呼吸正常化的可塑性机制尚不清楚。这些兴奋性颈动脉化学感受器效应的一种被提出的途径(Res.Plan，图1，第1页)是通过二阶孤束(NTS)神经元投射到面旁呼吸群/后梯形核(pFRG/RTN)。PHOX2B表达(PHOX2B)的pFRG/RTN神经元被认为：1)接受和整合多种兴奋性输入，包括来自颈动脉小体的那些；2)在睡眠中为前Bvtzinger复合体(PreBvtzzC)呼吸节律性神经元提供关键的兴奋性驱动；因此，PHOX2B神经元的功能障碍是中枢性和阻塞性睡眠呼吸暂停、化学感受器和运动事件反应异常的基础。另一种假设是，颈动脉的兴奋效应是通过改变脑干呼吸神经元的神经调节剂输入来实现的。我们将测试这些假设和其他假设，具体目的如下：1)确定PHOX2B pFRG/RTN神经元破坏对清醒和睡眠山羊呼吸的影响。假设：双侧破坏PHOX2B pFRG/RTN神经元将导致：a)清醒时呼吸不足，这将在NREM睡眠期间因长时间的呼吸暂停而加剧，以及b)减弱对高碳酸血症、低氧和运动的呼吸反应。这些假说的验证将支持pFRG/RTN神经元为呼吸提供关键的兴奋性输入的概念，特别是在睡眠期间。2)确定山羊CBD是否导致脑干呼吸网络兴奋性神经调节和抑制性神经调节之间的平衡发生中心转移。假设：CBD后，当山羊呼吸不足和CO2敏感性降低时，经PreBvtzC透析的模拟脑脊液(MCSF)中兴奋性和抑制性神经调节剂的浓度将从基线水平分别降低和升高。同样，在CBD后，将谷氨酸受体激动剂注射到前BvtzC的呼吸机反应将会降低。这些假说的验证将支持紧张性兴奋性颈动脉活动影响神经调节剂介导的呼吸节律性神经元兴奋性的概念。3)确定CBD后所观察到的时间依赖性可塑性(恢复)是否是通过兴奋性神经调节机制的上调。假设：在山羊CBD后两周，当他们不再呼吸不足时，流出的MCSF中通过PreBvtzC透析的兴奋性和抑制性神经调节剂的浓度以及向PreBvtzC注射谷氨酸受体激动剂的换气反应将达到或高于正常水平。此外，死后免疫组织化学将显示前BvtzC内具有兴奋性神经调节剂受体的神经元的百分比增加。这些假说的验证将与CBD后可塑性是由于呼吸网络内脑干兴奋性神经调节机制上调的概念一致。4)研究pFRG/RTN损毁后CBD对清醒和睡眠山羊呼吸的影响。假设：当山羊在切除pFRG/RTN一个月后进行CBD时，对呼吸的影响将小于CBD后的正常情况。这一假设的验证将表明颈动脉传入兴奋效应的中枢调节的主要途径(研究计划，图1，第1页)是通过pFRG/RTN。 公共卫生相关性： 数以百万计的美国人在睡眠期间，呼吸的循环机制不能正常工作，或者呼吸道崩溃，分别导致中枢性和阻塞性睡眠呼吸暂停。反复的呼吸暂停会导致体内的低氧气和高二氧化碳，这在许多情况下会导致动脉高血压、中风、心脏病发作、日间嗜睡、言语障碍和精神障碍。这些呼吸障碍和由此引起的疾病在退伍军人中非常突出，导致许多退伍军人的生活质量很差。在对山羊的拟议研究中，我们将消除大脑中调节呼吸的系统的一部分，我们假设这将导致睡眠期间呼吸紊乱。然而，我们预计到一个月后，呼吸将恢复正常。对脑细胞的尸检研究将提供对大脑如何从重大损伤中恢复的洞察。因此，本研究将是改善对患有睡眠呼吸障碍的退伍军人和在战斗中脑损伤的军人的护理和管理的又一步。

DESCRIPTION (provided by applicant): Neurons within the respiratory network regulate and coordinate respiratory pump and airway muscle activation. As such, the level of pulmonary ventilation is determined by the balance of excitatory and inhibitory inputs to these neurons. It has long been assumed that the primary excitatory drive to breathe is from intracranial CO2- H+ chemoreceptors. However, severe hypoventilation and reduced central CO2-H+ chemosensitivity after carotid body denervation (CBD) suggest a major excitatory drive is provided by carotid chemoreceptor afferents. It is unknown how, despite the presence of the intact and highly-sensitive intracranial chemoreceptors, CBD leads to hypoventilation, and furthermore what mechanisms of plasticity govern the normalization of breathing two or more weeks after CBD. One proposed pathway (Res. Plan, Figure 1, page 1) for these excitatory carotid chemoreceptor effects is through second order solitary tract (NTS) neuronal projections to the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN). Phox2b-expressing (Phox2b+) pFRG/RTN neurons are hypothesized to: 1) receive and integrate multiple excitatory inputs, including those from the carotid bodies, and 2) provide the critical excitatory drive to pre-Bvtzinger complex (preBvtzC) respiratory rhythmogenic neurons during sleep; hence, dysfunction of Phox2b+ neurons underlies central and obstructive sleep apnea, and abnormal chemoreceptor and exerciseventilatory responses. An alternative hypothesis is that the carotid excitatory effect is through changes in neuromodulator inputs to brainstem respiratory neurons. We will test these and other hypotheses with the following Specific Aims: 1) Determine the effects on breathing in awake and asleep goats of destruction of Phox2b+ pFRG/RTN neurons. Hypothesis: Bilateral destruction of Phox2b+ pFRG/RTN neurons will cause: a) hypoventilation while awake which will be accentuated during NREM sleep by prolonged apneas, and b) attenuated ventilatory responses to hypercapnia, hypoxia, and exercise. Validation of these hypotheses will support the concept that the pFRG/RTN neurons provide critical excitatory input for breathing, particularly during sleep. 2) Determine whether CBD in goats induces central shifts in the balance between excitatory and inhibitory neuromodulation of the brainstem respiratory network. Hypothesis: After CBD when goats hypoventilate and CO2 sensitivity is reduced, the concentration of excitatory and inhibitory neuromodulators in effluent mock cerebrospinal fluid (mCSF) dialyzed through the preBvtzC will be decreased and increased respectively from baseline. Also after CBD, the ventilatory response to an injection of a glutamate receptor agonist into the preBvtzC will be reduced. Validation of these hypotheses will support the concept that tonic excitatory carotid activity affects neuromodulator-mediated excitability of respiratory rhythmogenic neurons. 3) Determine whether the observed time-dependent plasticity (recovery) after CBD is through upregulation of excitatory neuromodulatory mechanisms. Hypothesis: Two weeks after CBD in goats when they are no longer hypoventilating, the concentrations of excitatory and inhibitory neuromodulators in effluent mCSF dialyzed through the preBvtzC and the ventilatory response to a glutamate receptor agonist injection into the preBvtzC will be at or above normal. Furthermore, post-mortem immunohistochemistry will show increased percentage of neurons with receptors for excitatory neuromodulators in the preBvtzC. Validation of these hypotheses will be consistent with the concept that plasticity after CBD is due to upregulation of brainstem excitatory neuromodulatory mechanisms within the respiratory network. 4) Determine the effects on breathing in awake and sleeping goats of CBD after pFRG/RTN lesions. Hypothesis: When goats undergo CBD a month after lesioning the pFRG/RTN, the effects on breathing will be less than what normally occurs after CBD. Validation of this hypothesis will indicate the major pathway (Res. Plan, Figure 1, page 1) for central mediation of carotid afferent excitatory effect is through the pFRG/RTN. PUBLIC HEALTH RELEVANCE: In millions of Americans during sleep, the cycling mechanism of breathing does not function properly or airways collapse resulting in central and obstructive sleep apnea respectively. The repeated apneas result in low O2 and high CO2 in the body which in many leads to arterial hypertension, strokes, heart attacks, daytime somnolence, dysphasia, and psychiatric disorders. These breathing disorders and the resultant diseases are prominent in the VA population leading to poor quality of life of many veterans. In the proposed studies on goats, we will eliminate part of the brain's system that regulates breathing which we hypothesize will result in disordered breathing during sleep. However, we expect that by a month later, breathing will have returned toward normal. Post mortem studies on brain cells will provide insight into how the brain recovers from major injury. Thus the present study will be another step toward improving care and management of veterans with sleep disordered breathing and also for military personal that had brain injury during combat.