Identifying Neural Substrates of Behavior in Drosophila Melanogaster

本研究的目标是利用神经活动的定向操纵技术来识别和功能定义特定行为背后的大脑网络。作为这种调查的模型，我们正在确定的网络，管理成年果蝇从蛹的情况下出现后不久执行的行为程序。这个程序包括一个适应性行为阶段，它介导寻找合适的环境，和一个先天阶段，它驱动翅膀的扩张，使它们适合飞行。阐明该程序的两个组件的电路的基础上，承诺一个详细的了解如何内在和外在因素的行为，单独和一致，招募运动模式组装行为序列。神经元网络相互作用和适应组织行为的机制的识别应该有助于阐明行为组织的失败，这是许多精神障碍的根源。 到目前为止，我们的工作集中在果蝇出现后行为序列的先天组成部分。这一部分由两个协调执行的运动模式组成，两者都由激素囊子控制。使用技术，允许有针对性地抑制特定的脑细胞在生活中，行为苍蝇，我们已经确定了两个功能不同的群体bursicon表达神经元。一组将囊子分泌到血淋巴（即血液）中，以影响翅膀水平的生理变化，而另一组在中枢神经系统内分泌囊子，以诱导负责翅膀扩张的运动模式。这两个群体是必要的机翼扩张。除了这两组，我们还确定了一组不表达囊子的调节神经元，但调节囊子表达组的激素释放。 我们的工作在很大程度上依赖于遗传技术，这些技术使我们能够操纵特定细胞亚群中的神经元活动。在过去，我们已经开发了用于组成性沉默（即UAS-EKO）和增强（即UAS-NaChBac）神经元电活动的工具。我们最近补充了这些工具，其中一个允许使用温度的小幅下降（即UAS-TRPM 8）的神经元的急性激活。这个工具利用了哺乳动物的冷敏感离子通道，最近使我们能够确定果蝇的囊子表达神经元不仅是翅膀扩张所必需的，而且也足以完成这一过程：囊子表达细胞的激活启动了整个翅膀扩张程序。这意味着一些或所有的bursicon表达神经元充当这个程序的命令神经元。 为了确定哪些囊体表达神经元可能充当命令神经元，我们需要更精细的技术来操纵体内单个细胞或细胞群的功能。为此，我们开发了组合的分裂Gal 4系统，其允许在感兴趣的细胞组内选择性地靶向基因。分裂Gal 4结合了来自酵母双杂交系统的技术，因为它将Gal 4分子分成其组分DNA结合（DBD）和转录激活（TA）结构域。每个结构域融合到两个互补的异二聚化亮氨酸拉链中的一个，使得DBD和TA结构域在表达两者的细胞中缔合。在这些细胞中，并且仅在这些细胞中，Gal 4转录活性重建。通过在体内独立靶向两个结构域，我们可以在表达两个结构域的细胞亚群中选择性地激活Gal 4s UAS结合位点下游的转基因。 我们已经利用了这个系统，通过靶向DBD结构域的囊子表达神经元和TA增强子陷阱线，表达Gal 4 TA（或更有效的TA的HSV-1 VP 16转录因子）在任意模式，包括不同的子集的囊子表达神经元。我们已经产生了允许UAS转基因在许多独特的囊体表达神经元亚群中表达的细胞系，目前正在分析选择性抑制或激活这些神经元的后果。 使用我们正在开发的广泛工具，对果蝇封闭后行为的神经元底物进行研究，应该可以深入了解所有神经系统产生和组织行为所使用的原则。此外，它应该作为一个电路映射方法的概念证明，可以扩展到哺乳动物行为的研究，类似的工具成为脊椎动物有机体。

The goal of this research is to exploit techniques for the targeted manipulation of neural activity to identify, and functionally define, brain networks underlying specific behaviors. As a model for such investigations, we are identifying the networks that govern the behavioral program executed by adult fruit flies shortly after emergence from the pupal case. This program consists of an adaptive behavioral phase, which mediates the search for a suitable environment, and an innate phase, which drives expansion of the wings to make them flight-worthy. Elucidation of the circuits underlying both components of this program promises a detailed understanding of how intrinsic and extrinsic factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Identification of the mechanisms by which neuronal networks interact and adapt to organize behavior should shed light on the failures in behavioral organization, which lie at the root of many mental disorders. Our work thus far has focused on the innate component of the post-emergence behavioral sequence in Drosophila. This component consists of two coordinately executed motor patterns, both of which are governed by the hormone bursicon. Using techniques that permit the targeted suppression of specific brain cells in living, behaving flies, we have identified two functionally distinct groups of bursicon-expressing neurons. One group secretes bursicon into the hemolymph (i.e. blood) to effect physiological changes at the level of the wing, while the other secretes bursicon within the central nervous system to induce the motor patterns responsible for wing expansion. Both groups are necessary for wing expansion. In addition to these two groups, we have also identified a group of regulatory neurons that do not express bursicon, but modulate the release of the hormone from the bursicon-expressing groups. Our work depends heavily on genetic techniques that allow us to manipulate neuronal activity in specific subsets of cells. In the past, we have developed tools for the constitutive silencing (i.e. UAS-EKO) and enhancement (i.e. UAS-NaChBac) of electrical activity in neurons. We have recently supplemented these tools with one that permits the acute activation of neurons using small decrements in temperature (i.e. UAS-TRPM8). This tool, which exploits a cold-sensitive ion channel from mammals, has recently allowed us to determine that the bursicon-expressing neurons of Drosophila are not only necessary for wing expansion, but are also sufficient for this process: Activation of the bursicon-expressing cells initiates the entire wing expansion program. This implies that some or all of the bursicon-expressing neurons act as command neurons for this program. To identify which bursicon-expressing neurons might act as command neurons, we have required more refined techniques for manipulating the function of individual cells, or groups of cells in vivo. To that end we developed the combinatorial, Split Gal4 system, which permits selective gene targeting within a cell group of interest. Split Gal4 incorporates technology from the yeast two-hybrid system in that it divides the Gal4 molecule into its component DNA-binding (DBD) and transcription activation (TA) domains. Each domain is fused to one of two complementary, heterodimerizing leucine zippers so that the DBD and TA domains associate in cells that express both. In these cells, and in these cells alone, is Gal4 transcriptional activity reconstituted. By independently targeting the two domains in vivo, we can activate transgenes downstream of Gal4s UAS binding site selectively in the subset of cells that expresses both domains. We have exploited this system by targeting the DBD domain to bursicon-expressing neurons and making TA enhancer trap lines that express the Gal4 TA (or the more potent TA of the HSV-1 VP16 transcription factor) in arbitrary patterns that include different subsets of the bursicon-expressing neurons. We have generated lines that permit expression of UAS-transgenes in numerous unique subsets of bursicon-expressing neurons and are currently analyzing the consequences of selectively suppressing or activating these neurons. Investigation of the neuronal substrates of posteclosion behavior in Drosophila using the broad palette of tools we are developing should provide insight into the principles used by all nervous systems to generate and organize behavior. In addition, it should serve as a proof of concept of a circuit mapping approach that can be extended to studies of mammalian behavior as similar tools become available for vertebrate organisms.