Real-time measurements of neurotransmission in Drosophila melanogaster

描述（由申请人提供）：本研究的长期目标是研究果蝇（Drosophila melanogaster）中枢神经系统中神经递质信号传导的动力学。本项目的具体目标是开发一种微电极方法来测量在完整的果蝇神经系统中5-羟色胺的实时释放和清除。果蝇是生物学家的一个流行模型系统，因为它很容易进行基因突变，并且与更高级的生物体具有同源性，包括类似的神经递质系统。苍蝇中枢神经系统的小尺寸（8 nL）已经排除了神经递质释放和摄取的实时研究。这项研究对人类健康有意义，因为血清素是一种重要的神经递质，其信号与精神疾病有关，包括抑郁症和药物滥用。然而，5-羟色胺浓度的调节机制还不清楚。具体目标是：1.描述果蝇幼虫个体的实时内源性5-羟色胺释放和摄取。带有蓝光敏感通道的转基因果蝇将被用来特异性地引发血清素的释放。这项技术将被用来测试可卡因管理的影响，对血清素转运体活性和遗传操作对神经递质释放的影响的假设。2.比较果蝇多巴胺能和多巴胺能信号的调控。重复的刺激将被用来评估合成和再循环在维持释放中的重要性。3.果蝇5-羟色胺转运体活性快速测定方法的建立。少量的血清素会被压力喷射到神经索中，然后在微电极上被检测到。该技术将允许测试关于药理学试剂或5-羟色胺转运蛋白突变对5-羟色胺信号传导的影响的假设。这些实验将导致对果蝇的检测，这对于筛选调节神经递质水平的遗传因子是有价值的，从而对神经传递的时间过程有更好的基本了解。

DESCRIPTION (provided by applicant): The long-term goal of this research is to study the dynamics of neurotransmitter signaling in the central nervous system of Drosophila melanogaster, the fruit fly. The specific goal of this project is to develop a microelectrode method to measure the real-time release and clearance of serotonin in an intact fruit fly nervous system. The fruit fly is a popular model system for biologists because of the ease of making genetic mutations and its homology with higher order organisms, including similar neurotransmitter systems. The small size of the fly central nervous system (8 nL) has precluded real-time studies of neurotransmitter release and uptake. This study has implications for human health because serotonin is an important neurotransmitter whose signaling is implicated in mental illnesses, including depression and drug abuse. However, the mechanisms for regulation of serotonin concentrations are not well understood. The specific aims are: 1. To characterize real-time, endogenous serotonin release and uptake in individual Drosophila larva. Genetically modified flies with a blue-light sensitive channel will be used to specifically elicit serotonin release. This technique will be used to test hypotheses about the effects of cocaine administration on serotonin transporter activity and the effects of genetic manipulations on neurotransmitter release. 2. To compare the regulation of serotonergic and dopaminergic signaling in Drosophila. Repeated stimulations will be used to assess the importance of synthesis and recycling in maintaining release. 3. Development of a rapid assay for serotonin transporter activity in Drosophila. Small amounts of serotonin will be pressure ejected into the nerve cord and then detected at the microelectrode. This technique will allow tests of hypotheses about the effect of pharmacological agents or mutations of the serotonin transporter on serotonin signaling. These experiments will result in assays for the fly that will be valuable for screening genetic factors that regulate neurotransmitter levels, resulting in a better fundamental understanding of the time course of neurotransmission.