Development of a Novel System to Study Cholinergic Synaptic Homeostasis

描述(由申请人提供)：突触稳态是神经元用来平衡全球神经活动变化的一种保护性机制。在过去的十年里，谷氨酸能突触在这一领域得到了深入的研究，然而，关于突触的稳态是否也由其他兴奋性受体介导，如烟碱型乙酰胆碱受体(NAChRs)，几乎一无所知。事实上，胆碱能突触动态平衡与阿尔茨海默病(AD)和尼古丁依赖等重要病理条件有关。在R21的应用中，我们的目标是验证一种研究胆碱能突触动态平衡的新系统，并在一个尚未探索的重要领域取得新的突破。果蝇中枢神经系统是果蝇中枢神经元的主要兴奋性受体，为研究胆碱能突触动态平衡提供了理想的模型。由于哺乳动物的α7nAChRs是nAChRs中最丰富和分布最广泛的，并与阿尔茨海默病、尼古丁成瘾、尼古丁诱导的癫痫和精神分裂症有关，因此我们将重点放在果蝇α7样受体(Dα7)上来验证我们的模型。使用原代神经元培养和培养的脑准备，我们建议操纵神经活动，并分析突触强度的变化以及nAChR定位和分布的变化。我们还将通过检测对Dα7nAChRs(巨型纤维介导的逃逸行为)介导的已知行为输出的影响，来测试胆碱能突触稳态的生理学相关性。这一新系统的开发，包括原代培养、体外脑准备和可量化的行为输出的组合，将是该领域独一无二的，并使我们能够回答期待已久的关于胆碱能突触动态平衡的问题。

DESCRIPTION (provided by applicant): Synaptic homeostasis is a protective mechanism employed by neurons to counterbalance changes in global neural activity. The last decade has seen intensive study in this field for glutamatergic synapses, however, almost nothing is known about whether synaptic homeostasis is also mediated by other excitatory receptors, such as nicotinic acetylcholine receptors (nAChRs). Indeed, cholinergic synaptic homeostasis has been implicated in important pathological conditions, such as Alzheimer's disease (AD) and nicotine dependence. In this R21 application, our goal is to validate a new system for studying cholinergic synaptic homeostasis and break new ground in an important area that has not yet been explored. The Drosophila CNS provides an ideal model for studying cholinergic synaptic homeostasis since nAChRs are the major excitatory receptor in Drosophila central neurons. Since mammalian α7 nAChRs are among the most abundant and widespread of nAChRs, and have been implicated in Alzheimer's disease, nicotine addiction, nicotine-induced seizures, and schizophrenia, we focus on the Drosophila α7 like receptor (Dα7) to validate our model. Using primary neuronal cultures in parallel with a cultured brain preparation, we propose to manipulate neural activity, and assay for changes in synaptic strength and changes in nAChR localization and distribution. We will also test the physiological relevance of cholinergic synaptic homeostasis by examining effects on a known behavioral output mediated by Dα7 nAChRs, the giant fiber mediated escape behavior. The development of this novel system, which involves the combination of primary cultures, an ex-vivo brain preparation, and a quantifiable behavioral output, will be unique to the field and allow us to answer long-awaited questions about cholinergic synaptic homeostasis.