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

最近的研究表明，通过暴露于尼古丁，nAChR的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。在这项资助中，我们建议在果蝇中解决这个问题，这是一个简单的，遗传上可访问的模型系统。我们最近已经证明，nAChRs，箭毒和α-BTX阻断，介导培养的果蝇神经元之间的快速兴奋性传输的主要形式，提供了一个独特的机会，探索重要的遗传和生化信号调节这些受体和传输在中央胆碱能突触。我们的初步实验将集中在表征的特性的全细胞电流诱导急性暴露培养的野生型神经元的不同浓度的尼古丁。第二个具体目标将检查尼古丁在调节功能性nAChRs中的作用。通过监测尼古丁诱导、乙酰胆碱诱导和胆碱能兴奋性突触后电流（EPSC）的电生理学特性，评估这些受体生物物理特性的潜在变化。为了确定受体数量是否发生变化，将在暴露于尼古丁之前和之后监测125 l-alphaBTX结合。在第三个目标中，我们将研究短时间重复暴露于尼古丁的影响，模仿吸烟期间发生的暴露，对nAChR表达的影响。最后，我们将研究尼古丁诱导的电流的性质和调节的nAChRs的神经元从傻瓜，一个学习和记忆突变体与缺陷的cAMP信号。dunce和野生型神经元之间的nAChRs和胆碱能突触电流的电生理学和药理学特性的差异将是重要的，以确定尼古丁是否激活相同的cAMP信号级联反应，这是重要的强化学习模式在苍蝇。我们的研究结果将为尼古丁诱导nAChRs调控的分子机制提供重要的见解。这些数据也将有助于普遍了解的分子机制，重要的调节兴奋性突触，可能是高度保守的脊椎动物和无脊椎动物之间的传输，因此应有助于设计药物和/或治疗，旨在调节可塑性突触通路有助于成瘾的人。

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.