Chronic nicotine and synaptic transmission in brainstem respiratory neurons

脑干呼吸神经元的慢性尼古丁和突触传递

• 批准号: 8508277
• 负责人: Ralph Frank Fregosi
• 金额: $ 29.6万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-10 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508277
• 关键词: 3-Dimensional; AMPA Receptors; Acetylcholine; Action Potentials; Affect; Age; Agonist; Anatomy; Apnea; Asthma; Autonomic nervous system disorders; Bathing; Blood - brain barrier anatomy; Brain Stem; Brain region; Breathing; Cardiac; Cardiovascular Abnormalities; Cell Nucleus; Cells; Cellular Morphology; Chemicals; Chest wall structure; Child; Childhood; Cholinergic Receptors; Chronic; Clinical Research; Complex; Confocal Microscopy; Control Animal; Control Groups; Coupled; Data; Deglutition; Deglutition Disorders; Dendrites; Development; Differentiation and Growth; Dyes; Electrophysiology (science); Exhibits; Exposure to; Frequencies; Glutamates; Glycine; Glycine Receptors; Goals; Growth; Growth and Development function; Health; Hour; Human; Image; Immunohistochemistry; In Vitro; Incidence; Infant; Laboratories; Lead; Learning; Location; Maps; Mastication; Measurement; Measures; Mediating; Memory; Morphology; Motor; Motor Neurons; Muscle; N-Methyl-D-Aspartate Receptors; Neonatal; Nerve; Nervous system structure; Neurons; Neurotransmitter Receptor; Neurotransmitters; Nicotine; Nicotine Dependence; Nicotinic Receptors; Obstructive Sleep Apnea; Personal Satisfaction; Phenotype; Positioning Attribute; Pregnant Women; Presynaptic Receptors; Proteins; Rattus; Reflex control; Regulation; Research Design; Resistance; Resolution; Risk; Saline; Series; Shapes; Signal Transduction; Sleep Disorders; Slice; Smoke; Speech Delay; Structure; Sudden infant death syndrome; Synapses; Synaptic Transmission; Techniques; Testing; Tetrodotoxin; Tobacco smoke; Tongue; Trees; Voltage-Clamp Technics; Work; behavior measurement; density; desensitization; design; feeding; gamma-Aminobutyric Acid; in utero; in vivo; infancy; neonate; neuromuscular; neuronal cell body; neurotransmitter release; offspring; patch clamp; postsynaptic; pressure; presynaptic; pup; receptor; reconstruction; research study; respiratory; response; rib bone structure; sleep abnormalities; sucking; synaptogenesis; transmission process; voltage clamp

DESCRIPTION (provided by applicant): At least 20% of pregnant women smoke, and their offspring have a higher than normal incidence of impaired cardiac function, autonomic nervous system disorders, sleep disorders, delayed speech and central and obstructive apneas. Importantly, recent studies document that the main respiratory phenotype in nicotine-exposed human neonates is a higher incidence of obstructive sleep apnea. It is widely accepted that obstructive apnea is caused largely by abnormal activation of tongue muscles, which are in turn controlled by hypoglossal motoneurons. Work in our laboratory beginning in 2003-2004 shows that in utero and early neonatal nicotine exposure (developmental nicotine exposure, DNE) leads to complex changes in breathing and hypoglossal motoneuron structure and function, including: a) desensitization of nAChRs; b) reduced excitatory synaptic input; c) increased input resistance, suggesting that the neurons are smaller; d) altered neuronal responses to inhibitory and excitatory agonists, including nicotine; e) altered ventilatory control in vivo, including increased apnea duration, with the entire apneic period associated with the loss of tongue muscle activity. Here we propose a series of studies designed to systematically examine the effects of DNE on both presynaptic and postsynaptic regulation of hypoglossal motoneuron function, motoneuron morphology, including estimates of the distribution of glutamatergic and GABAergic synapses upon motoneurons, and control of the tongue musculature in vivo. Specific Aim 1 tests the hypothesis that DNE reduces the release of both excitatory and inhibitory neurotransmitters from glutamatergic, GABAergic and glycinergic neurons in the vicinity of the hypoglossal motoneurons, using whole cell voltage clamp techniques. Aim 2 is designed to determine how DNE exaggerates the post-synaptic response of hypoglossal motoneurons to agonists of GABAA, glycine, NMDA and AMPA receptors. These post-synaptic effects will be evaluated by blocking presynaptic input to hypoglossal motoneurons, and studying postsynaptic effects by injecting small volumes of receptor agonists, while measuring changes in whole cell current and conductance under voltage clamp. Aim 3 tests the hypothesis that DNE disrupts the normal signals that regulate dendritic growth and synapse formation, leading to a reduction in the number of glutamatergic and GABAergic synapses formed upon the hypoglossal motoneurons. This hypothesis will be tested by filling motoneurons with dyes, and using 3-dimensional confocal microscopy to reconstruct the motoneuron cell body and dendritic tree, followed by detailed measures of somatic and dendritic anatomy. These data will be coupled with immunohistochemistry to examine the distribution of glutamatergic and GABAergic synapses that impinge upon the motoneurons, and how the number, position and density of these synapses change with DNE. Aim 4 examines the very real consequences of DNE by testing the hypothesis that DNE leads to an increased frequency and duration of obstructive, central and mixed apneas in vivo, due to reduced tongue muscle activation and diminished neuromuscular responses to changes in upper airway pressure. For these studies we will use lightly anesthetized neonatal rat pups wherein measurements of rib cage expansion and the EMG activity of inspiratory intercostal and tongue muscles are recorded. We will measure the frequency and duration of central, obstructive and mixed apneas, and the genioglossus EMG before, during and after each apneic episode. Reflex control of tongue muscles evoked by changing upper airway pressure will also be measured and quantified. All experiments will be done in neonatal rat pups exposed to either nicotine (experimental group) or saline (control group) in utero. These studies are clinically important because DNE in human infants is associated with an abnormally high incidence of breathing, feeding, swallowing and cardiovascular abnormalities that affects the health and well-being of millions of human infants in infancy and childhood. It is therefore crucial to begin establishing the mechanisms that lead to abnormal development of the brainstem neurons that regulate these critical homeostatic functions.

描述（由申请人提供）：至少20%的孕妇吸烟，其后代心脏功能受损、自主神经系统疾病、睡眠障碍、言语延迟以及中枢性和阻塞性呼吸暂停的发生率高于正常。重要的是，最近的研究表明，尼古丁暴露的人类新生儿的主要呼吸表型是阻塞性睡眠呼吸暂停的发生率较高。目前普遍认为阻塞性呼吸暂停主要是由舌肌的异常激活引起的，而舌肌又受舌下神经运动神经元的控制。我们实验室从2003-2004年开始的工作表明，在子宫内和新生儿早期接触尼古丁（发育性尼古丁暴露，DNE）导致呼吸和舌下运动神经元结构和功能的复杂变化，包括：a）nAChR的脱敏; B）兴奋性突触输入减少; c）输入阻力增加，表明神经元较小; d）对抑制性和兴奋性激动剂（包括尼古丁）的神经元反应改变; e）体内呼吸控制改变，包括呼吸暂停持续时间增加，整个呼吸暂停期与舌肌活动丧失相关。在这里，我们提出了一系列的研究，旨在系统地检查DNE的舌下神经运动神经元功能，运动神经元形态，包括估计的分布上的突触和GABA能突触的突触的突触前和突触后调节的影响，并在体内舌肌肉组织的控制。具体目的1使用全细胞电压钳技术测试DNE减少舌下运动神经元附近的谷氨酸能、GABA能和甘氨酸能神经元的兴奋性和抑制性神经递质的释放的假设。目的2研究DNE如何增强舌下神经运动神经元对GABAA、甘氨酸、NMDA和AMPA受体激动剂的突触后反应。这些突触后效应将通过阻断舌下神经运动神经元的突触前输入进行评价，并通过注射小体积的受体激动剂研究突触后效应，同时在电压钳下测量全细胞电流和电导的变化。目的3验证DNE干扰调节树突生长和突触形成的正常信号，导致舌下神经运动神经元上形成的谷氨酸能和GABA能突触数量减少的假设。这一假设将通过填充运动神经元与染料，并使用三维共聚焦显微镜重建运动神经元细胞体和树突树，然后通过体细胞和树突解剖的详细措施进行测试。这些数据将与免疫组织化学相结合，以检查影响运动神经元的多巴胺能和GABA能突触的分布，以及这些突触的数量、位置和密度如何随着DNE而变化。目的4通过测试DNE导致体内阻塞性、中枢性和混合性呼吸暂停的频率和持续时间增加的假设来检查DNE的非常真实的后果，这是由于舌肌激活减少和对上气道压力变化的神经肌肉反应减少。对于这些研究，我们将使用轻度麻醉的新生大鼠幼仔，其中记录胸廓扩张的测量值以及吸气肋间和舌肌的EMG活动。我们将测量中枢性、阻塞性和混合性呼吸暂停的频率和持续时间，以及每次呼吸暂停发作之前、期间和之后的颏舌肌肌电图。还将测量和量化由改变上气道压力引起的舌肌反射控制。所有实验均在子宫内暴露于尼古丁（实验组）或盐水（对照组）的新生大鼠幼仔中进行。这些研究在临床上很重要，因为人类婴儿的DNE与呼吸、喂养、吞咽和心血管异常的异常高发生率相关，这些异常影响数百万婴儿和儿童的健康和福祉。因此，必须开始建立机制， 调节这些重要的自我平衡功能的脑干神经元发育异常。