PILOT STUDY--NICOTINE ROLE IN REGULATION OF NACHR IN DROSOPHILA

试点研究--尼古丁在果蝇 NACHR 调节中的作用

• 批准号: 6495109
• 负责人: DIANE K O'DOWD
• 金额: $ 17.92万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6495109
• 关键词: Drosophilidae; chemical stimulation; cholinergic receptors; cyclic AMP; drug addiction; electrodes; electrophysiology; neural transmission; neurons; neurophysiology; nicotine; receptor expression; synapses; tissue /cell culture; tobacco abuse

Recent studies suggest that modifications in the number and function of nAChRs, by exposure to nicotine, plays an important role in the process of addiction. However, the molecular mechanisms important in regulating these receptors in the mammalian CNS remain poorly understood. In this grant we propose to address this question in Drosophila, a simple, genetically accessible model system. We have recently demonstrated that nAChRs, blocked by both curare and alpha-BTX, mediate the primary form of fast excitatory transmission between cultured Drosophila neurons, providing a unique opportunity to explore the genetic and biochemical signals important in regulation of these receptors and transmission at central cholinergic synapses. Our initial experiments will focus on characterization of the properties of the whole cell currents induced by acute exposure of cultured wildtype neurons to various concentration of nicotine. The second specific aim will examine the role of nicotine in regulation of the-functional nAChRs. Potential changes in the biophysical properties of these receptors will be assessed by monitoring the electrophysiological properties of nicotine-induced, ACh-induced, and cholinergic excitatory postsynaptic currents (EPSCs), following chronic exposure to nicotine. To determine if there are changes in the numbers of receptors, 125l-alphaBTX binding will be monitored before and after exposure to nicotine. In the third aim we will examine the effects of short duration, repetitive exposure to nicotine, mimicking the exposure that occurs during smoking, on nAChR expression. Finally, we will examine the properties of nicotine induced currents and regulation of nAChRs in neurons from dunce, a learning and memory mutant with defects in CAMP-signaling. Differences in the electrophysiological and pharmacological properties of the nAChRs and cholinergic synaptic currents between dunce and wildtype neurons will be important in determining if nicotine activates the same CAMP-signaling cascade that is important in reinforced learning paradigms in flies. The results of our studies will provide important insights into the molecular mechanisms underlying nicotine induced regulation of nAChRs. These data will also contribute to the general understanding of the molecular mechanisms important in regulating transmission at excitatory synapses, likely to be highly conserved between vertebrates and invertebrates, and therefore should aid in the design of drugs and/or therapies aimed at modulating plasticity in synaptic pathways contributing to addiction in humans.

最近的研究表明，通过接触尼古丁而改变 nAChR 的数量和功能，在成瘾过程中发挥着重要作用。然而，对于调节哺乳动物中枢神经系统中这些受体的重要分子机制仍然知之甚少。在这笔拨款中，我们建议在果蝇中解决这个问题，果蝇是一个简单的、遗传上可访问的模型系统。我们最近证明，被箭毒和 α-BTX 阻断的 nAChR 介导培养的果蝇神经元之间快速兴奋性传递的主要形式，为探索在调节这些受体和中枢胆碱能突触传递中重要的遗传和生化信号提供了独特的机会。我们最初的实验将集中于表征由培养的野生型神经元急性暴露于不同浓度的尼古丁所诱导的全细胞电流的特性。第二个具体目标是研究尼古丁在调节功能性 nAChR 中的作用。这些受体生物物理特性的潜在变化将通过监测长期接触尼古丁后尼古丁诱导的、乙酰胆碱诱导的和胆碱能兴奋性突触后电流（EPSC）的电生理特性来评估。为了确定受体数量是否发生变化，将在接触尼古丁之前和之后监测 125l-alphaBTX 结合。在第三个目标中，我们将研究短期、重复接触尼古丁（模仿吸烟期间发生的接触）对 nAChR 表达的影响。最后，我们将研究尼古丁诱导电流的特性以及 dunce 神经元中 nAChR 的调节，dunce 是一种具有 CAMP 信号传导缺陷的学习和记忆突变体。笨蛋和野生型神经元之间 nAChR 和胆碱能突触电流的电生理和药理学特性的差异对于确定尼古丁是否激活相同的 CAMP 信号级联非常重要，这对于果蝇的强化学习范例很重要。我们的研究结果将为尼古丁诱导的 nAChR 调节的分子机制提供重要的见解。这些数据还将有助于对调节兴奋性突触传递重要的分子机制的一般理解，这些机制可能在脊椎动物和无脊椎动物之间高度保守，因此应有助于设计旨在调节导致人类成瘾的突触途径可塑性的药物和/或疗法。