Identifying Neural Substrates of Behavior in Drosophila

识别果蝇行为的神经基础

• 批准号: 7136784
• 负责人: Benjamin H White
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7136784
• 关键词: Drosophilidae; animal genetic material tag; behavioral /social science research tag; behavioral genetics; brain electrical activity; brain mapping; ethology; gene expression; gene targeting; genetically modified animals; green fluorescent proteins; hormone regulation /control mechanism; limb movement; metamorphosis; neural inhibition; neural transmission; neurohormones; neurons; neuropeptides; neuropsychology; neuroregulation; serotonin; technology /technique development

The goal of this research program is to exploit, and further develop, techniques for manipulating neural activity to identify the brain circuits underlying specific behaviors. Using the Gal4-UAS gene targeting system of Drosophila melanogaster to drive the expression of genes whose products inhibit neuronal excitability, we are selectively suppressing the activity of subsets of neurons and analyzing the effects of this manipulation on behavior. We are particularly interested in the suite of hormonally coordinated and developmentally programmed behaviors executed by the adult fly shortly after emergence from the pupal case, with an immediate focus on those necessary for wing expansion. To identify neuronal substrates of these behaviors we use two approaches: one in which defined subsets of neurons, and the other in which random subsets, are inhibited (using Gal4 lines with defined promoters and Gal4 enhancer-trap lines, respectively). In each case, patterns of suppression that affect the behaviors of interest are identified for further characterization. Using the first approach, we have found that inhibition of electrical activity in a small subset of neurons that express the neuromodulator CCAP suppresses wing expansion in flies. Using the second approach, we have identified 27 patterns of suppression (i.e. enhancer-trap lines) that generate wing expansion deficits. Using a transgenic line that specifically suppresses Gal4 activity in CCAP-expressing neurons (CCAP-Gal80), we have demonstrated that 26 of these enhancer-trap lines exert their effects on wing expansion by acting within the CCAP neurons. One of these lines expresses primarily in a subset of CCAP-expressing neurons that also expresses the hormone bursicon, which is known from genetic studies to be required for wing expansion. We used this line to demonstrate that there are two functionally distinct groups of CCAP-expressing neurons. One group (the output group) is responsible for secreting bursicon into the hemolymph (blood); the other group (the regulatory group) modulates the activity of the output group. Having established that the CCAP-expressing neurons form a network that controls bursicon release, our goal is to provide a complete functional characterization of this network. On-going work with other enhancer-trap lines is designed to establish the functional identities of further subsets of CCAP-expressing neurons. To facilitate investigation of the CCAP network, we have developed two techniques that should be generally applicable to the targeted manipulation of neuronal function. The first technique permits the targeted enhancement of excitability using the gene encoding the bacterial sodium channel, NaChBac, fused to Green Fluorescent Protein (GFP). We have shown that NaChBac-GFP enhances excitability in Drosophila muscles and neurons and have used it to demonstrate that enhanced excitability in the regulatory group of CCAP-expressing neurons disrupts bursicon secretion. The second tool we have developed is a modification of the Gal4-UAS technique that incorporates technology from the yeast two-hybrid system. We have split the Gal4 molecule into its component DNA-binding (DB) and transcription activation (TA) domains and fused them to heterodimerizing leucine zippers. This permits the DB and TA domains to associate in cells that express both domains and reconstitute Gal4 transcriptional activity. Cells expressing a single domain lack this activity. By independently targeting the two domains in vivo, we can activate UAS transgenes selectively in the subset of cells that expresses both domains. This has allowed us to rationally restrict our manipulations of neural function to specific subsets of the CCAP network. We anticipate that this tool will find broad use in the refined targeting of genetic manipulations to small subsets of cells. Investigation of the neuronal substrates of posteclosion behavior in Drosophila using the broad palette of tools we are developing should serve as a ?proof of concept? of a circuit mapping approach that can later be extended to studies of mammalian behavior.

这项研究计划的目标是开发并进一步发展操纵神经活动的技术，以识别特定行为背后的大脑回路。利用黑胃果蝇Gal4-UAS基因靶向系统来驱动其产物抑制神经元兴奋性的基因表达，我们有选择地抑制神经元亚群的活动，并分析这种操作对行为的影响。我们特别感兴趣的是成虫从蛹中羽化后不久所执行的一系列激素协调和发育程序行为，并立即关注那些翅膀扩张所必需的行为。为了确定这些行为的神经元底物，我们使用了两种方法：一种是定义神经元子集，另一种是随机子集被抑制（分别使用具有定义启动子的Gal4系和Gal4增强子陷阱系）。在每种情况下，影响感兴趣的行为的抑制模式被确定为进一步的表征。使用第一种方法，我们发现在表达神经调节剂CCAP的一小部分神经元中抑制电活动抑制了苍蝇的翅膀扩张。使用第二种方法，我们已经确定了27种抑制模式（即增强-陷阱线），产生机翼扩张缺陷。利用转基因系特异性抑制表达CCAP神经元中的Gal4活性（CCAP- gal80），我们证明了26个增强诱捕系通过在CCAP神经元中发挥作用来影响翅膀的扩张。其中一种细胞系主要在表达ccap的神经元子集中表达，该子集也表达囊囊激素，从遗传学研究中得知，囊囊激素是翅膀扩张所必需的。我们用这条线来证明有两组功能不同的ccap表达神经元。一组（输出组）负责将滑囊分泌到血淋巴中；另一组（调节组）调节输出组的活动。在确定表达ccap的神经元形成控制滑囊释放的网络后，我们的目标是提供该网络的完整功能表征。正在进行的与其他增强子陷阱细胞系的合作旨在确定进一步表达ccap的神经元亚群的功能身份。