Brainstem nAChR Subtypes & Their Effect on GI Function

脑干 nAChR 亚型

• 批准号: 6727710
• 负责人: Richard Alan Gillis
• 金额: $ 23.81万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727710
• 关键词: autoradiography; brain stem; fluorescent in situ hybridization; gastrointestinal motility /pressure; genetically modified animals; laboratory mouse; laboratory rat; microinjections; neuropharmacology; nicotine; nicotinic receptors; receptor expression; voltage /patch clamp

DESCRIPTION (provided by applicant): Administration of nicotine and/or smoking has been shown to have important effects on gastric function. Evidence of others and our own data strongly indicate that these effects are due to nicotine acting on neuronal nicotinic acetylcholine receptors (nAChRs) in the dorsal motor nucleus of the vagus (DMV) and medial subnucleus of the tractus solitarius (mNTS). The goals of our proposed studies are to: (i) investigate the influence of DMV and mNTS nAChRs in the effects of systemically administered nicotine on gastric function and (ii) to use DMV and mNTS nAChRs as model systems to learn more about native CNS nicotinic receptors, their nature, their function and their regulation. The Specific Aims of our research are to: (i) determine the subunit composition of nAChRs in the rat DMV and mNTS that mediate the effects of nicotine on gastric tone and motility; (ii) examine regulation of the function and density of nAChRs in rat DMV and mNTS after chronic administration of nAChR-stimulating doses of nicotine, and the agonist-induced responsiveness of nAChRs in the DMV and mNTS after acute exposure to sub-activating doses of nicotine; and (iii) test the hypothesis that nAChRs observed to exist in the DMV and mNTS as revealed by pursuing Specific Aims 1 & 2 play an important role in the gastric effects (DMV and mNTS) and cardiovascular effects (mNTS) of systemically administered nicotine. To achieve these Specific Aims, a focused but multidisciplinary approach will be used consisting of the following techniques: (i) pharmacological tools applied either in vivo by microinjection of drugs into the DMV and mNTS of anesthetized rats while monitoring gastric tone and motility, or in vitro to rat brain slice preparations containing the DMV and mNTS while monitoring electrophysiological events using the whole-cell patch-clamp recording method; (ii) in vitro autoradiography taking advantage of the high affinity epibatidine binding to nAChRs combined with selective denervation of efferent and afferent vagal fibers; (iii) immunofluorescent identification of nAChR subtypes in DMV and mNTS neurons; (v) fluorescent in situ hybridization technique to identify nAChR subunits associated with DMV neurons projecting to the fundus and antrum, (vi) knockout mice, in which one gene of interest is silenced, and the contribution of a particular nAChR subunit (e.g., a7 subunit) in eliciting a specific gastric response can be assessed. Findings made to date suggest that: (I) there are five different nAChRs in the DMV and mNTS that influence gastric function; (ii) there might be a viscerotopic organization of nAChR subunits on DMV motoneurons. That is a7 containing nAChRs might be located primarily on DMV neurons projecting to the fundus but not on DMV neurons projecting to the antrum; and (iii) iv. administered nicotine might act in the mNTS on a ct4B2-nAChR subtype to inhibit tonic contraction of the fundus. These and future data obtained from pursuing our Specific Aims will extend current understanding of the different receptor mediated effects of nicotine on parasympathetic nervous system control of the upper GI tract.

描述（由申请人提供）： 尼古丁和/或吸烟的管理已被证明具有重要的 对胃功能的影响其他人和我们自己的数据的有力证据 表明这些作用是由于尼古丁作用于神经元烟碱， 迷走神经背侧运动核（DMV）中的乙酰胆碱受体（nAChRs） 和孤束内侧亚核（mNTS）。我们的目标 建议的研究是：（i）调查DMV和mNTS nAChRs的影响 全身给予尼古丁对胃功能的影响， (ii)使用DMV和mNTS nAChR作为模型系统，以了解更多关于天然CNS的信息 烟碱受体，它们的性质，它们的功能和它们的调节。的 我们的研究的具体目的是：（i）确定 大鼠DMV和mNTS中的nAChRs介导尼古丁对胃粘膜的影响 张力和运动;（ii）检查调节的功能和密度， 慢性给予nAChR刺激后大鼠DMV和mNTS中的nAChR 尼古丁剂量，以及DMV中nAChR的激动剂诱导的反应性 和mNTS急性暴露于亚活化剂量的尼古丁后;和（iii） 测试假设，即观察到存在于DMV和mNTS中的nAChR， 通过追求特定目标1和2揭示了在胃中发挥重要作用， 全身性的作用（DMV和mNTS）和心血管作用（mNTS） 服用尼古丁。为了实现这些具体目标，一个有重点的，但 将采用多学科方法，包括以下技术： (i)通过显微注射药物在体内应用的药理学工具 进入麻醉大鼠的DMV和mNTS，同时监测胃张力， 运动，或在体外对含有DMV的大鼠脑切片制备物， mNTS，同时使用全细胞监测电生理事件 膜片钳记录方法;（ii）体外放射自显影， 结合nAChR的高亲和力地棘蛙素与选择性地棘蛙素结合， 传出和传入迷走神经纤维的去神经支配;（iii）免疫荧光 DMV和mNTS神经元中nAChR亚型的鉴定;（v）荧光标记， 应用原位杂交技术鉴定DMV相关nAChR亚单位 投射到胃底和胃窦的神经元，（vi）敲除小鼠，其中一个 感兴趣的基因被沉默，并且特定的nAChR的贡献 子单元（例如，a7亚基）在引发特异性胃反应中的作用， 评估。研究结果表明：（一）有五种不同的 DMV和mNTS中影响胃功能的nAChR;（ii）可能存在 DMV运动神经元上nAChR亚单位的内脏组织。这是A7 含有nAChRs的神经元可能主要位于DMV神经元上， 在胃底但不在投射到胃窦的DMV神经元上;和（iii）iv. 给予的尼古丁可能在mNTS中作用于ct 4 B2-nAChR亚型， 眼底的紧张性收缩。这些和未来的数据获得的追求 我们的具体目标将扩展目前对不同受体的理解， 尼古丁对副交感神经系统控制的介导作用 上消化道