Chronic nicotine and synaptic transmission in brainstem respiratory neurons

描述(申请人提供)：至少20%的孕妇吸烟，她们的子女心脏功能受损、自主神经系统障碍、睡眠障碍、言语迟缓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，最近的研究证明，暴露于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生率较高。人们普遍认为，阻塞性呼吸暂停主要是由舌下运动神经元控制的舌肌异常激活引起的。我们实验室从2003-2004年开始的工作表明，在宫内和新生儿早期暴露尼古丁(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)nAChRs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)神经元对包括尼古丁在内的抑制性和兴奋性激动剂的反应发生变化；e)体内呼吸控制发生变化，包括呼吸暂停持续时间延长，整个呼吸暂停期与肌肉活动的丧失有关。在这里，我们提出了一系列的研究，旨在系统地研究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，包括对谷氨酸和GABA能突触在运动神经元上的分布的估计，以及体内舌肌的控制。具体目的1利用全细胞电压钳技术，验证DNE可减少舌下运动神经元附近谷氨酸、GABA能和甘氨酸能神经元兴奋性和抑制性神经递质的释放。目的2研究DNE如何夸大舌下运动神经元对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突触前传入舌下运动神经元，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来评估。目的3验证DNE干扰调节树突生长和突触形成的正常信号，导致舌下运动神经元上形成的谷氨酸和GABA能突触数量减少的假说。这一假设将通过在运动神经元中填充染料，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来验证，然后对体细胞和树突进行详细的解剖测量。这些数据将与免疫组织化学结合起来，研究冲击运动神经元的谷氨酸和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的变化而变化。目的通过检验DNE在体内导致阻塞性、中枢性和混合性呼吸暂停的频率和持续时间增加的假说来检验DNE的真实后果，这是由于舌肌活动减少和神经肌肉对上呼吸道压力变化的反应减弱所致。在这些研究中，我们将使用轻微麻醉的新生大鼠幼鼠，在这些幼鼠中，记录胸腔扩张以及吸气、肋间和舌肌的肌电活动。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，以及在每一次呼吸暂停发作之前、期间和之后的颧舌肌肌电。还将测量和量化因改变上呼吸道压力而引起的舌肌反射控制。所有实验都将在子宫内暴露于尼古丁(实验组)或生理盐水(对照组)的新生大鼠幼鼠身上进行。这些研究具有重要的临床意义，因为人类婴儿中的DNE与呼吸、进食、吞咽和心血管异常的异常高发生率有关，这些异常影响了数百万婴儿在婴儿期和儿童期的健康和福祉。因此，至关重要的是开始建立导致 脑干神经元的异常发育，调节这些关键的体内平衡功能。

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.