Hybrid atomic force-optical imaging system to investigate prenatal nicotine

描述（由申请人提供）：神经元烟碱乙酰胆碱受体（nAchR）是神经递质乙酰胆碱和外源胆碱能配体（例如香烟烟雾中的尼古丁）的靶标。烟碱受体在外周（颈动脉体）和中枢（脑干神经元）结构上表达，参与介导缺氧的呼吸反应。产前尼古丁会破坏呼吸稳定性，减弱对缺氧应激的代偿性呼吸反应，并导致婴儿猝死综合症 (SIDS)。对尼古丁暴露的适应不良呼吸反应的一方面似乎是由呼吸节律形成的中枢 GABA 能调节的减少介导的。这部分是由于 nAchR 亚基的“功能丧失”造成的。该提案的总体目标是开发一种新颖的成像系统，该系统结合了原子力显微镜（AFM）和高速光学成像，以研究尼古丁对缺氧反应的呼吸节律的发展和稳定性的影响。我们建议使用这种混合成像系统来确定产前尼古丁是否会改变负责产生呼吸节律的中枢呼吸回路中的突触传递。具体目标是： 1. 使用光学成像识别尼古丁引起的中枢呼吸网络活动时空模式的变化。根据初步研究，我们假设产前接触尼古丁会改变缺氧刺激期间中枢呼吸活动的空间和时间模式，并降低自动复苏的能力。 2. 使用原子力显微镜评估尼古丁对 17 和 14 nAchR 结合特性的影响。根据初步研究，我们假设产前尼古丁会降低中枢呼吸神经元表达的含有 nAchR 的 17 和 14 亚基的结合概率。从该提案中获得的结果具有重要意义，因为它们将是第一个使用保留脑桥延髓呼吸回路的制剂直接可视化尼古丁诱导的中枢呼吸神经元关键群体神经元活动时空模式变化的结果。该提案中概述的实验还将为产前尼古丁对 nAchR 功能结合特性的有害影响提供新的见解。这些新发现可能有助于制定临床建议，以减轻与 SIDS 和其他产前呼吸系统疾病的发生相关的缺氧引起的呼吸抑制。公共卫生相关性：产前接触香烟烟雾中的尼古丁会使婴儿容易患婴儿猝死综合症 (SIDS)。最近的科学证据强烈表明，尼古丁可能会导致控制呼吸的脑细胞上的烟碱受体过度激活，从而使婴儿容易患上 SIDS。该提案的目标是确定产前尼古丁暴露对大脑控制呼吸的影响。了解尼古丁对呼吸神经元的影响非常重要，因为 SIDS 的发病机制尚未完全了解，因此严重限制了潜在的药理学靶点和治疗策略。这也具有临床意义，因为孕妇的尼古丁替代疗法通常被认为是戒烟计划中的安全替代疗法。

DESCRIPTION (provided by applicant): Neuronal nicotinic acetylcholine receptors (nAchR) are targets for the neurotransmitter acetylcholine and exogenous cholinergic ligands such as nicotine from cigarette smoke. Nicotinic receptors are expressed on peripheral (carotid body) and central (brainstem neurons) structures that are involved in mediating the respiratory responses to hypoxia. Prenatal nicotine destabilizes breathing, diminishes the compensatory respiratory response to hypoxic stress and contributes to sudden infant death syndrome (SIDS). One aspect of the maladaptive respiratory response to nicotine exposure appears to be mediated by a reduction in central GABAergic regulation of respiratory rhythm formation. This results, in part, from the "loss-of-function" of nAchR subunits. The overall goal of this proposal is to develop a novel imaging system which combines atomic force microscopy (AFM) and high-speed optical imaging to investigate the effect of nicotine on the development and stability of breathing rhythms in response to hypoxia. We propose to use this hybrid imaging system to determine whether prenatal nicotine alters synaptic transmission in central respiratory circuits responsible for generating breathing rhythms. The specific aims are: 1. Identify nicotine-induced changes the spatiotemporal patterns of central respiratory network activity using optical imaging. Based on Preliminary studies, we hypothesize that prenatal exposure to nicotine will alter the spatial and temporal patterns of central respiratory activity during hypoxic stimulation and will reduce the ability to autoresuscitate. 2. Evaluate the effect of nicotine on the binding properties of 17 and 14 nAchRs using atomic force microscopy. Based on Preliminary studies, we hypothesize that prenatal nicotine will reduce the binding probability of 17 and 14 subunit containing nAchR expressed on central respiratory neurons. Results obtained from this proposal are significant because they will be the first to directly visualize nicotine-induced changes in the spatiotemporal patterns of neuronal activity in key populations of central respiratory neurons using a preparation that preserves pontomedullary respiratory circuitry. Experiments outlined in this proposal will also provide new insight to the deleterious affects of prenatal nicotine on the functional binding properties of nAchRs. These new findings may aid in developing clinical recommendations to alleviate hypoxic-induced respiratory depression associated with the incidence of SIDS and other prenatal respiratory disorders. PUBLIC HEALTH RELEVANCE: Prenatal exposure to nicotine from cigarette smoke predisposes infants to sudden infant death syndrome (SIDS). Recent scientific evidence strongly suggests that nicotine may predispose infants to SIDS by causing excessive activation of nicotinic receptors on brain cells that control breathing. The goal of this proposal is to determine the effect of prenatal nicotine exposure on the control of breathing by the brain. Knowledge of the effects of nicotine on respiratory neurons is important since the pathogenesis of SIDS remains incompletely understood, and thus, severely limits potential pharmacological targets and treatment strategies. It is also of clinical significance since nicotine replacement therapy for pregnant women is often regarded as a safe alternative in smoking cessation programs.