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

最近的研究表明，尼古丁对nAChRs数量和功能的改变在成瘾过程中起着重要作用。然而，在哺乳动物中枢神经系统中调节这些受体的重要分子机制仍然知之甚少。在这项拨款中，我们建议在果蝇身上解决这个问题，这是一个简单的、可通过基因获得的模型系统。我们最近已经证明，被Curare和α-BTX阻断的nAChRs，介导了培养的果蝇神经元之间快速兴奋性传递的初级形式，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。我们的初步实验将集中在描述由培养的野生型神经元急性暴露于不同浓度的尼古丁所引起的全细胞电流的特性。第二个具体目标将研究尼古丁在调节非功能性nAChRs中的作用。这些受体生物物理特性的潜在变化将通过监测尼古丁诱导的、ACh诱导的和长期接触尼古丁后的胆碱能兴奋性突触后电流(EpSCs)的电生理特性来评估。为了确定受体数量是否有变化，将监测暴露于尼古丁前后的125l-alphaBTX结合情况。在第三个目标中，我们将研究短期、重复暴露于尼古丁的影响，模拟吸烟期间的暴露，对nAChR表达的影响。最后，我们将研究尼古丁诱导电流的特性和Dunce神经元中nAChRs的调节。Dunce是一种学习和记忆突变体，在cAMP信号转导方面存在缺陷。NAChRs和胆碱能突触电流在愚型神经元和野生型神经元之间的电生理和药理学特性的差异将在确定尼古丁是否激活相同的cAMP信号级联反应方面具有重要意义，而cAMP信号级联反应在增强果蝇的学习范例中是重要的。我们的研究结果将为尼古丁诱导nAChRs调节的分子机制提供重要的见解。这些数据还将有助于全面理解在调节兴奋性突触传递方面重要的分子机制，这些机制可能在脊椎动物和无脊椎动物之间高度保守，因此应该有助于设计旨在调节导致人类成瘾的突触通路可塑性的药物和/或疗法。

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.