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

最近的研究表明，尼古丁对nachr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，果蝇是一个简单的，遗传上可获得的模型系统。我们最近发现，被curare和α - btx阻断的nachr介导了培养的果蝇神经元之间快速兴奋传递的主要形式，这为探索这些受体调控和中枢胆碱能突触传递的重要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在对培养的野生型神经元急性暴露于不同浓度尼古丁诱导的全细胞电流特性的表征上。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尼古丁诱导、乙酰胆碱诱导和胆碱能兴奋性突触后电流（EPSCs）在慢性尼古丁暴露后的电生理特性来评估。为了确定受体的数量是否有变化，125l-alphaBTX结合将在暴露于尼古丁前后进行监测。在第三个目标中，我们将研究短期重复暴露于尼古丁对nAChR表达的影响，模拟吸烟期间的暴露。最后，我们将研究尼古丁诱导电流的特性和dunce神经元中nachr的调节，dunce是一种具有camp信号缺陷的学习和记忆突变体。在愚型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将是确定尼古丁是否激活在果蝇强化学习范式中重要的相同的camp信号级联的重要因素。我们的研究结果将为尼古丁诱导的nachr调控的分子机制提供重要的见解。这些数据也将有助于对兴奋性突触传输调节的重要分子机制的一般理解，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.