Identifying Neural Substrates of Behavior in Drosophila Melanogaster

昆虫蜕皮序列代表了一个简单的，强大的，易于处理的模型，用于研究神经调节机制，管理行为。由于蜕皮序列的启动涉及行为优先级的深刻转变，因此对这些序列的研究提供了了解支配行为状态变化的神经调节机制的机会。此外，由于蜕皮行为是固有的顺序，他们允许系统的调查如何运动程序组装和连续执行的神经系统。最后，蜕皮序列的研究有望深入了解保守的电路如何被重新配置，以产生非常不同的行为。例如，在果蝇中，蛹和成虫蜕皮前和蜕皮后的运动序列几乎没有相似之处，尽管它们受到一组共同的神经调节/激素输入的控制。通过类比计算，这些输入可以被视为用高级编程语言编写的指令，然后编译成不同的电机输出模式。揭示蜕皮中神经编译的机制可能会深刻地告知我们神经调节剂如何通过重新配置神经网络的活动来促进神经计算的理解。 为了研究这些问题，我们的实验室试图阐明电路，管理蛹和成人蜕皮序列在果蝇，虽然在过去的一年中，我们主要集中在蛹蜕皮的神经元决定因素。如上所述，蛹蜕皮严格依赖于内在因素，这一事实从以下观察中最明显：将外周释放的激素蜕皮触发激素（ETH）外源性应用于分离的蛹神经系统足以触发表达控制蜕皮的其他关键神经调节剂的神经元的顺序激活。证明这种顺序激活与广义虚构蜕皮序列的产生相关是我们目前工作的目标，我们在过去一年的努力集中在识别和表征ETH的直接神经元靶点上。为了鉴定这些靶标，我们使用了我们实验室在前一年的一篇主要论文中介绍的特洛伊外显子技术（Diao et al.，2015，Cell Reports 10，14101421）。特洛伊外显子能够识别和遗传操纵表达特定感兴趣基因的神经元，我们已经使用这种工具选择性地靶向表达ETH受体/基因的两种亚型ETHRA和ETHRB中的每一种的神经元。 正如今年发表的一篇论文所描述的（Diao et al.，2016，Genetics 202：175-189），我们已经表明ETHRA和ETHRB在很大程度上不同的神经元亚群中表达，并且ETHRA-而不是ETHRB-表达神经元是所有发育阶段的蜕皮所需的。我们的遗传和神经元操作的结果表明，ETHRB在蛹和成人，但不是幼虫蜕皮的重要作用，他们进一步证实了在蛹蜕皮的ETHRA表达神经元，共表达激素甲壳类动物促性腺激素肽（CCAP）的要求。在这项工作中，我们还确定了其他功能重要的ETHRA表达神经元的子集，包括一个共同表达肽Leucokinin。我们证明，白细胞激肽表达神经元调节液体平衡，以促进蜕皮在蛹阶段。 因此，我们最近的工作增强了我们对ETH下游神经元介导蛹蜕皮序列的理解。如前所述，这些神经元包括表达其他激素的细胞，这些激素对产生蛹蜕皮序列至关重要，如CCAP。CCAP和其他激素的神经元靶点的鉴定和功能表征是目前实验室工作的目标。这项工作也依赖于特洛伊外显子的方法，我们成功地应用这种技术来阐明蛹蜕皮电路说明了一个一般的战略映射神经调节网络。使用受体基因作为遗传和神经元操作的切入点，可以在网络中不同水平的神经元之间建立功能连接模式。 总之，在过去的一年里，我们在阐明果蝇神经系统中支持蜕皮的发育动态回路方面取得了明显的进展。我们的工作继续揭示蜕皮行为的神经元底物，并补充了果蝇神经系统功能结构的其他研究。正如去年发表的一份特邀观点所指出的那样（白色，B.H.，2016，J Neurogenet：30：54-61），对果蝇和其他遗传模型动物的神经系统组织的深入研究有望全面和前所未有地了解神经系统如何在一生中仲裁动物的需求和欲望，并应提供对所有神经系统的一般运作的不可或缺的见解。因此，这些见解应该有助于阐明行为组织的缺陷，这些缺陷是许多精神障碍的根源。

Insect ecdysis sequences represent a simple, robust, and tractable model for studying the neuromodulatory mechanisms that govern behavior. Because initiation of an ecdysis sequence involves a profound shift in behavioral priorities, study of these sequences offers the opportunity to understand the neuromodulatory mechanisms that govern changes in behavioral state. In addition, because ecdysis behaviors are inherently sequential, they permit the systematic investigation of how motor programs are assembled and serially executed by the nervous system. Finally, the study of ecdysis sequences promises insight into how conserved circuits can be variably configured to generate immensely different behaviors. In Drosophila, for example, the motor sequences performed at pupal and adult ecdysis before and after metamorphosis, respectively are scarcely similar though they are governed by a common set of neuromodulatory/hormonal inputs. By analogy to computing, these inputs can be regarded as instructions written in a higher programming language that are then compiled into different motor output patterns. Exposing the mechanisms of neural compilation in ecdysis is likely to deeply inform our understanding of how neuromodulators contribute to neurocomputation by reconfiguring the activity of neural networks. To investigate these questions, our laboratory seeks to elucidate the circuitry that governs both the pupal and adult ecdysis sequences in Drosophila, though during the past year, we have focused primarily on the neuronal determinants of pupal ecdysis. As noted above, pupal ecdysis is strictly dependent on intrinsic factors, a fact most evident from the observation that exogenous application of the peripherally released hormone, Ecdysis Triggering Hormone (ETH) to an isolated pupal nervous system is sufficient to trigger sequential activation of neurons that express other key neuromodulators governing ecdysis. Demonstrating that this sequential activation correlates with the generation of a generalized fictive ecdysis sequence is a goal of our current work, and our efforts over the last year have focused on identifying and characterizing the direct neuronal targets of ETH. To identify these targets, we have used the Trojan exon technique introduced by our laboratory in a major paper from the previous year (Diao et al., 2015, Cell Reports 10, 14101421). Trojan exons enable the identification and genetic manipulation of neurons that express a specific gene of interest and we have used this tool to selectively target neurons that express each of the two isoforms of the ETH receptor/ gene, ETHRA and ETHRB. As described in a paper published this year (Diao et al., 2016, Genetics 202:175-189), we have shown that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. Our results from both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval ecdysis, and they further confirm the requirement at pupal ecdysis for ETHRA-expressing neurons that co-express the hormone Crustacean Cardioactive Peptide (CCAP). In this work, we also identified other functionally important subsets of ETHRA-expressing neurons including one that co-expresses the peptide Leucokinin. We demonstrated that Leucokinin-expressing neurons regulate fluid balance to facilitate ecdysis at the pupal stage. Our recent work thus augments our understanding of neurons that act downstream of ETH to mediate the pupal ecdysis sequence. As noted, these neurons include cells that express other hormones critical for generating the pupal ecdysis sequence, such as CCAP. Identification and functional characterization of the neuronal targets of CCAP and other hormones is the goal of current work in the laboratory. This work also relies on the Trojan exon method and our success in applying this technique to elucidating the pupal ecdysis circuit illustrates a general strategy for mapping hormonally regulated networks. Using a receptor gene as an entry point for genetic and neuronal manipulations, one can establish patterns of functional connectivity between neurons at different levels in the network. In summary, we have made clear progress during the last year in elucidating the developmentally dynamic circuit in the Drosophila nervous system that supports ecdysis. Our work continues to uncover neuronal substrates of ecdysis behavior and complements other research on the functional architecture of the Drosophila nervous system. As argued in an invited perspective published during the last year (White, B.H., 2016, J Neurogenet: 30:54-61), intensive investigation of nervous system organization in Drosophila and other genetic model animals promises a comprehensive and hitherto unprecedented understanding of how nervous systems function to arbitrate an animals needs and desires in the course of a lifetime and should deliver indispensable insights into the general operation of all nervous systems. These insights should thus shed light on the deficits in behavioral organization that lie at the root of many mental disorders.