Chronic Nicotine Effects on Receptor Subtypes

尼古丁对受体亚型的慢性影响

• 批准号: 6821385
• 负责人: DAVID C PERRY
• 金额: $ 29.23万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821385
• 关键词: autoradiography; brain mapping; immunochemistry; immunoprecipitation; laboratory rat; neuropharmacology; neurotransmitter transport; nicotine; nicotinic receptors; protein structure function; psychopharmacology; radiotracer; receptor binding; receptor expression; western blottings

DESCRIPTION (provided by applicant): Prolonged exposure to nicotine causes profound alterations in nicotine receptors (nAChR), including upregulation and changes in functional status. While in vitro studies show clear differences in these responses among different nAChR subtypes, little is known about differential subtype responses after chronic nicotine exposure in vivo, such as occurs with use of tobacco products or nicotine replacement therapy. If the numbers and activity of some nAChR subtypes is altered more than other subtypes, this would change the pharmacological spectrum of activity and behavioral responses to nicotine; therefore knowledge of subtype-selective responses is essential for understanding nicotine neurobiology. The overarching goal of this proposal is to determine the consequences of chronic in vivo nicotine exposure on expression and function of nAChR subtypes, including alpha4-beta2, alpha-beta4 and alpha6/alpha3-beta2. Chronic drug exposure will be achieved by perfusion of rats with osmotic mini-pumps; it will be compared to acute exposure by injection. Aim 1 will employ a new method of quantitative autoradiography developed in our lab, using [1251] epibatidinewith subtype-selective competitors, that allows measurement of receptor subtypes in small regions, to assess effects of chronic nicotine exposure and recovery from such exposure. We predict that alpha4-beta2 receptors will be much more sensitive to upregulation, and recover more slowly. We will also test the effects of acute nicotine, to test whether effects seen are due to length of time of exposure. We will then use both homogenate binding and autoradiography with more selective radioligands to confirm any changes detected with [125l] epibatidine autoradiography. Aim 2 will use quantitative immunochemical techniques with subunit-selective antibodies to provide important confirmation and extension of any binding changes detected in Aim 1. Aims 3 and 4 will use two different methods to correlate binding changes to functional activity of nAChR subtypes; as above, we hypothesize that activity of alpha4-beta 2 receptors will be disproportionately affected by chronic nicotine. Functional changes caused by chronic (and acute) nicotine and recovery will be directly determined in multiple brain regions by measuring 86Rb efflux (Aim 3). Functional effects of treatments at the systems level will be assessed by measuring nicotinic-stimulated release of different neurotransmitters in selected regions (Aim 4). For these two aims, subtype selectivity will be obtained by the use of selective agonists and antagonists. The systematic application of these different methods to measure numbers and function of nAChR subtypes will allow us to develop a much more complete picture of the brain's response to chronic stimulation by this important drug of abuse.

描述（由申请人提供）： 长期暴露于尼古丁会引起尼古丁受体（nAChR）的深刻变化，包括上调和功能状态的变化。虽然体外研究显示不同nAChR亚型之间的这些反应存在明显差异，但对体内慢性尼古丁暴露后的差异亚型反应知之甚少，例如使用烟草产品或尼古丁替代疗法。如果一些nAChR亚型的数量和活性比其他亚型改变更多，这将改变尼古丁的药理学活性谱和行为反应;因此，了解亚型选择性反应对于理解尼古丁神经生物学至关重要。本提案的总体目标是确定长期体内尼古丁暴露对nAChR亚型（包括α 4-β 2、α-β 4和α 6/α 3-β 2）表达和功能的影响。将通过用渗透微型泵灌注大鼠实现慢性药物暴露;将其与通过注射的急性暴露进行比较。目标1将采用我们实验室开发的一种新的定量放射自显影方法，使用[1251] epibatidine与亚型选择性竞争剂，可以测量小区域的受体亚型，以评估慢性尼古丁暴露的影响和从这种暴露中恢复。我们预测，α 4-β 2受体将对上调更加敏感，并且恢复得更慢。我们还将测试急性尼古丁的影响，以测试所看到的影响是否是由于暴露时间的长度。然后，我们将使用匀浆结合和放射自显影与更具选择性的放射性配体，以确认任何变化检测与[125 l] epibatidine放射自显影。目标2将使用亚基选择性抗体的定量免疫化学技术，为目标1中检测到的任何结合变化提供重要的确认和扩展。目标3和4将使用两种不同的方法将结合变化与nAChR亚型的功能活性相关联;如上所述，我们假设α 4-β 2受体的活性将不成比例地受到慢性尼古丁的影响。慢性（和急性）尼古丁引起的功能变化和恢复将通过测量86 Rb流出直接确定多个脑区（目的3）。将通过测量所选区域中尼古丁刺激的不同神经递质释放来评估系统水平治疗的功能效应（目标4）。为了这两个目的，将通过使用选择性激动剂和拮抗剂来获得亚型选择性。系统地应用这些不同的方法来测量nAChR亚型的数量和功能，将使我们能够更全面地了解大脑对这种重要药物滥用的慢性刺激的反应。