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性胆碱能配体（如香烟烟雾中的尼古丁）的靶点。烟碱受体在外周（颈动脉体）和中枢（脑干神经元）结构上表达，参与介导对缺氧的呼吸反应。产前尼古丁会使呼吸不稳定，减少对缺氧应激的代偿性呼吸反应，并导致婴儿猝死综合征（SIDS）。对尼古丁暴露的适应不良呼吸反应的一个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”所致。该提案的总体目标是开发一种新型成像系统，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时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，以确定是否产前尼古丁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.利用光学成像技术识别尼古丁引起的中枢呼吸网络活动时空模式的变化。基于初步研究，我们假设产前暴露于尼古丁将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价尼古丁对17和14 nAchR结合特性的影响。基于初步研究，我们推测产前尼古丁会降低中枢呼吸神经元上表达的含17和14亚基的nAchR的结合概率。从这个建议获得的结果是显着的，因为他们将是第一个直接可视化尼古丁诱导的变化的时空模式的神经元活动的关键群体的中枢呼吸神经元使用的准备，保留pontomedullary呼吸回路。本提案中概述的实验也将为产前尼古丁对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发病率相关的缺氧诱导的呼吸抑制。公共卫生相关性：产前暴露于香烟烟雾中的尼古丁易使婴儿患婴儿猝死综合征（SIDS）。最近的科学证据有力地表明，尼古丁可能通过过度激活控制呼吸的脑细胞上的尼古丁受体而使婴儿易患SIDS。这项提案的目的是确定产前尼古丁暴露对大脑呼吸控制的影响。尼古丁对呼吸神经元的影响的知识是重要的，因为SIDS的发病机制仍然不完全清楚，因此，严重限制了潜在的药理学靶点和治疗策略。它也具有临床意义，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方案。

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.