Real-time measurements of neurotransmission in Drosophilia melanogaster

 描述(由申请人提供)：模型系统用于了解神经传递是如何调节的，以及在疾病期间它是如何发生故障的。果蝇是一种很有吸引力的模式生物，因为它很容易遗传改变，繁殖快，价格便宜，行为复杂。果蝇有许多类似哺乳动物的神经递质系统，包括多巴胺和5-羟色胺能系统。虽然已经在果蝇幼虫的腹神经索上测量了多巴胺和5-羟色胺，但在成年果蝇的大脑中还没有测量到内源性释放。成年果蝇的神经系统发育完全，有望成为研究精神卫生疾病中神经递质调节的良好模型系统。我的实验室的长期目标是开发新的分析工具，以了解生物系统中的实时神经传递。本项目的目的是研究成虫和幼虫特定神经束中多巴胺和5-羟色胺信号的调节。具体地说，我们将确定哪些5-羟色胺受体作为自身受体，以及环指蛋白11(RNF11)是如何调节多巴胺的，RNF11是NF-B途径的调节器。核心假设是，神经递质的调节随年龄和神经纤维的不同而不同。目的1是比较幼虫和成虫中枢神经系统不同部位的多巴胺释放。将通过光学激活基因插入的CsChrimson来激发释放，并将在成人中研究衰老对多巴胺释放的影响。在目标2中，我们将通过RNF11的RNAi敲除来探索RNF11对多巴胺的调节。D2自身受体拮抗剂抗精神病药物的作用将在RNF11 RNAi果蝇中进行研究，以调查RNF11对多巴胺自身受体调节的影响程度。这将是第一次研究RNF11对任何物种中的多巴胺信号的影响。在目标3中，我们将研究在幼虫(前脑和VNC)和成虫(蘑菇体，VNC)中刺激的5-羟色胺释放和再摄取。5-羟色胺系统是许多精神疾病治疗的靶标，因此在果蝇中了解它将使其成为精神疾病的模式生物。最后，对于目标4，我们将确定5-羟色胺自身受体对5-羟色胺的影响。 使用药理学和遗传学方法对幼虫和成虫进行调节。这项工作的预期结果是更好地表征特定果蝇神经管中的神经传递，并了解果蝇发育过程中多巴胺和5-羟色胺释放的调节。这项研究意义重大，因为它将提供神经递质信号的基础知识，以便使用果蝇可用的强大遗传工具来研究特定基因在控制神经传递中的作用。未来的研究可能会检测多巴胺和5-羟色胺能神经传递如何控制成年果蝇的疾病行为或功能。

 DESCRIPTION (provided by applicant): Model systems are used to understand how neurotransmission is regulated and how it malfunctions during diseases. Drosophila, the fruit fly, is an attractive model organism because it is easy to genetically alter, fast to breed, inexpensive, and has complex behaviors. Drosophila has many neurotransmitter systems similar to mammals, including dopaminergic and serotonergic systems. While dopamine and serotonin measurements have been made in the larval Drosophila ventral nerve cord, no measurements of endogenous release have been made in the adult fly brain. Adult flies have a fully developed nervous system and would be a good model system for studying the regulation of neurotransmitters in mental health diseases. The long-term goal of my lab is to develop new analytical tools to understand real-time neurotransmission in biological systems. The goal of this project is to characterize the regulation of dopamine and serotonin signaling in specific neuropil of the adult and larval Drosophila. Specifically, we will determine which serotonin receptors act as auto receptors and how dopamine is regulated by RING finger protein 11 (RNF11), a modulator of the NF-B pathway. The central hypothesis is that neurotransmitter regulation varies with age and by neuropil. Aim 1 is to compare dopamine release in discrete areas of the larval and adult CNS. Release will be evoked by optically activating genetically-inserted CsChrimson and the effect of aging on dopamine release will be studied in the adult. In Aim 2, we will explore the regulation of dopamine by RNF11 using RNAi knockdown of RNF11. The effect of D2 auto receptor antagonist antipsychotics will be studied in RNF11 RNAi flies, to investigate the extent to which RNF11 affects auto receptor regulation of dopamine. These will be the first studies of the effect of RNF11 on dopamine signaling in any species. In Aim 3, we will investigate stimulated serotonin release and reuptake in larvae (protocerebrum and VNC) and adults (mushroom bodies, VNC). The serotonin system is a target of many treatments for mental illness, so understanding it in flies will enable its use as a model organism of mental diseases. Finally, for Aim 4, we will determine the effect of serotonin auto receptors on serotonin regulation in larvae and adults using both pharmacological and genetic approaches. The expected outcomes of this work are better methods for characterizing neurotransmission in specific Drosophila neuropil and an understanding of the regulation of dopamine and serotonin release throughout Drosophila development. The research is significant because it will provide foundational knowledge of neurotransmitter signaling necessary to use the powerful genetic tools available for Drosophila to study the effects of specific genes in controlling neurotransmission. Future studies could examine how dopaminergic and serotonergic neurotransmission control behaviors or function in diseases in adult Drosophila.