Identifying Neural Substrates of Behavior in Drosophila

这项研究计划的目标是利用和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。使用果蝇的Gal 4-UAS基因靶向系统来驱动其产物抑制神经元兴奋性的基因的表达，我们选择性地抑制神经元子集的活性，并分析这种操作对行为的影响。我们特别感兴趣的是一套由成年苍蝇从蛹的情况下出现后不久执行的协调和发育程序化的行为，立即关注那些必要的翅膀扩张。为了识别这些行为的神经元底物，我们使用两种方法：一种是定义的神经元子集，另一种是随机子集，被抑制（分别使用具有定义的启动子的Gal 4系和Gal 4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识别用于进一步表征。使用第一种方法，我们发现抑制表达神经调节剂CCAP的一小部分神经元的电活动会抑制苍蝇翅膀的扩张。使用第二种方法，我们已经确定了27个模式的抑制（即增强陷阱线），产生机翼扩张赤字。使用一个转基因株系，特别是抑制Gal 4的活性在CCAP表达神经元（CCAP-Gal 80），我们已经证明，26这些增强子-陷阱线发挥其影响翼扩展内的CCAP神经元。其中一个细胞系主要在一个表达CCAP的神经元亚群中表达，该亚群还表达激素bursicon，这是遗传研究中已知的翅膀扩张所需的。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）负责将囊子分泌到血淋巴（血液）中;另一组（调节组）调节输出组的活性。已经确定CCAP表达神经元形成控制囊子释放的网络，我们的目标是提供该网络的完整功能表征。正在进行的工作与其他增强子陷阱线的目的是建立进一步的子集的CCAP表达神经元的功能特性。 为了便于调查的CCAP网络，我们已经开发了两种技术，应普遍适用于有针对性的操纵神经元功能。第一种技术允许使用与绿色荧光蛋白（GFP）融合的编码细菌钠通道NaChBac的基因靶向增强兴奋性。我们已经表明，NaChBac-GFP增强了果蝇肌肉和神经元的兴奋性，并用它来证明，增强的兴奋性的调节组的CCAP表达的神经元破坏囊分泌。我们开发的第二个工具是Gal 4-UAS技术的修改，该技术结合了酵母双杂交系统的技术。我们已经将Gal 4分子分裂成其组成部分DNA结合（DB）和转录激活（TA）结构域，并将它们融合到异源二聚化亮氨酸拉链上。这允许DB和TA结构域在表达这两个结构域的细胞中缔合并重建Gal 4转录活性。表达单一结构域的细胞缺乏这种活性。通过在体内独立靶向这两个结构域，我们可以在表达这两个结构域的细胞亚群中选择性地激活UAS转基因。这使我们能够合理地将神经功能的操作限制在CCAP网络的特定子集上。我们预计，该工具将在精细靶向小细胞亚群的遗传操作中得到广泛应用。调查的神经基板的posteclosion行为在果蝇使用广泛的调色板的工具，我们正在开发应作为一个？概念验证？这是一种电路映射方法，以后可以扩展到哺乳动物行为的研究。

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.