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: 8371126
• 负责人: Ralph Frank Fregosi
• 金额: $ 31.19万
• 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/8371126
• 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. PUBLIC HEALTH RELEVANCE: Our goal is to better understand how exposure to nicotine in utero and in the early neonatal period (Developmental Nicotine Exposure, DNE) alters development of nervous system structure and function in infants and young children, with specific focus on the hypoglossal motoneurons, which are critical for normal breathing, swallowing, sucking and chewing. Clinical studies have shown that DNE can lead to learning and memory problems, a greater rate of nicotine addiction, growth abnormalities, feeding and swallowing disorders, sleep abnormalities, and an increased risk of asthma, sudden infant death syndrome (SIDS) and both central and obstructive sleep apnea. Over the last several years, our laboratory has produced considerable evidence showing that DNE neonates exhibit a variety of abnormal anatomic and functional changes that adversely affect normal breathing control. We seek to understand the cellular mechanisms that cause these developmental changes. Here we propose a series of detailed experiments that will examine how DNE alters the structure and function of hypoglossal motor neurons, and correlate them with behavioral measures in neonatal rats studied in vivo. Hypoglossal motor neurons, located in the brainstem, control muscles of the tongue. Altered control of the tongue muscles is the major cause of breathing problems, including obstructive sleep apnea, as well as swallowing, sucking and chewing abnormalities. Moreover, because these neurons are large and readily accessible in slices of the neonatal rat brainstem, that produces a ventilatory rhythm; detailed in vitro studies of their structure and function are possible. We will determine how DNE influences the normal release of neurotransmitters onto the motoneurons; the function of inhibitory and excitatory neurotransmitter receptors on the neurons; the location and number of synapses; and the growth and development of dendrites. The in vivo experiments will allow us to determine how DNE influences the frequency and duration of central and obstructive apnea, and the reflex control of the tongue muscles. Techniques used include patch clamp electrophysiology of single hypoglossal motor neurons, immunohistochemistry, measures of neuron size and shape, and measures of tongue muscle activity and breathing in anesthetized neonatal rats. For all studies, neurons from DNE neonates will be compared to age-matched, saline-exposed control animals.

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、自主神经系统紊乱、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水平。重要的是，最近的研究表明，接触尼古丁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病率较高。人们普遍认为，阻塞性呼吸暂停主要是由舌肌异常激活引起的，而舌肌又由舌下运动神经元控制。我们实验室从 2003-2004 年开始的工作表明，子宫内和新生儿早期尼古丁暴露（发育性尼古丁暴露，DNE）会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 脱敏； b) 兴奋性突触输入减少； c) 输入电阻增加，表明神经元更小； d) 改变神经元对抑制性和兴奋性激动剂（包括尼古丁）的反应； e) 体内通气控制改变，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，我们提出了一系列研究，旨在系统地检查 DNE 对舌下运动神经元功能、运动神经元形态的突触前和突触后调节的影响，包括估计谷氨酸能和 GABA 能突触在运动神经元上的分布，以及体内舌头肌肉组织的控制。具体目标 1 测试了以下假设：DNE 使用全细胞电压钳技术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释放兴奋性和抑制性神经递质。目标 2 旨在确定 DNE 如何增强舌下运动神经元对 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运动神经元的突触前输入来评估，并通过注射少量受体激动剂来研究突触后效应，同时测量电压钳下全细胞电流和电导的变化。目标 3 测试了以下假设：DNE 破坏了调节树突生长和突触形成的正常信号，导致舌下运动神经元上形成的谷氨酸能和 GABA 能突触数量减少。该假设将通过用染料填充运动神经元，并使用 3 维共聚焦显微镜重建运动神经元细胞体和树突树，然后对体细胞和树突解剖结构进行详细测量来进行测试。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谷氨酸能和 GABA 能突触的分布，以及这些突触的数量、位置和密度如何随 DNE 变化。目标 4 通过检验 DNE 导致体内阻塞性呼吸暂停、中枢性呼吸暂停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 的真实后果，这是由于舌肌激活减少和对上呼吸道压力变化的神经肌肉反应减弱。对于这些研究，我们将使用轻度麻醉的新生大鼠幼崽，其中记录胸腔扩张的测量值以及吸气肋间肌和舌肌的肌电图活动。我们将测量中枢性呼吸暂停、阻塞性呼吸暂停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。通过改变上呼吸道压力引起的舌头肌肉的反射控制也将被测量和量化。所有实验都将在子宫内暴露于尼古丁（实验组）或盐水（对照组）的新生大鼠幼崽中进行。这些研究具有重要的临床意义，因为人类婴儿的 DNE 与异常高的呼吸、喂养、吞咽和心血管异常发生率有关，影响了数百万婴儿和儿童时期的健康和福祉。因此，至关重要的是开始建立导致 调节这些关键稳态功能的脑干神经元发育异常。 公共健康相关性：我们的目标是更好地了解子宫内和新生儿早期接触尼古丁（发育性尼古丁暴露，DNE）如何改变婴儿和幼儿神经系统结构和功能的发育，特别关注舌下运动神经元，这对于正常呼吸、吞咽、吸吮和咀嚼至关重要。临床研究表明，DNE 可导致学习和记忆问题、尼古丁成瘾、生长异常、喂养和吞咽障碍、睡眠异常以及哮喘、婴儿猝死综合症 (SIDS) 和中枢神经系统疾病的风险增加。阻塞性睡眠呼吸暂停。在过去的几年里，我们的实验室已经提供了大量证据，表明 DNE 新生儿表现出各种异常的解剖和功能变化，对正常呼吸控制产生不利影响。我们寻求了解导致这些发育变化的细胞机制。在这里，我们提出了一系列详细的实验，将检查 DNE 如何改变舌下运动神经元的结构和功能，并将其与体内研究的新生大鼠的行为测量相关联。舌下运动神经元位于脑干，控制舌头的肌肉。舌头肌肉的控制改变是呼吸问题的主要原因，包括阻塞性睡眠呼吸暂停，以及吞咽、吸吮和咀嚼异常。此外，由于这些神经元很大，并且在新生大鼠脑干切片中很容易接触到，从而产生通气节律；对其结构和功能进行详细的体外研究是可能的。我们将确定 DNE 如何影响神经递质向运动神经元的正常释放；抑制性和兴奋性神经递质受体对神经元的功能；突触的位置和数量；以及树突的生长和发育。体内实验将使我们能够确定 DNE 如何影响中枢性和阻塞性呼吸暂停的频率和持续时间，以及舌肌的反射控制。使用的技术包括单个舌下运动神经元的膜片钳电生理学、免疫组织化学、神经元大小和形状的测量以及麻醉新生大鼠舌肌活动和呼吸的测量。对于所有研究，DNE 新生儿的神经元将与年龄匹配、暴露于盐水的对照动物进行比较。