Identifying Neural Substrates of Behavior in Drosophila Melanogaster

昆虫蜕皮序列为研究支配行为的神经调节机制提供了一个简单、健壮和易处理的模型。由于蜕皮序列的启动涉及行为优先顺序的深刻变化，因此对这些序列的研究提供了理解支配行为状态变化的神经调节机制的机会。此外，由于蜕皮行为本质上是连续的，它们允许对神经系统如何组装和连续执行运动程序进行系统研究。最后，对蜕皮序列的研究有助于洞察守恒的电路如何被可变地配置以产生非常不同的行为。例如，在果蝇中，分别在变态前后的蛹和成虫蜕皮时执行的运动序列几乎不相似，尽管它们受到一组共同的神经调节/激素输入的支配。类似于计算，这些输入可以被认为是用更高级编程语言编写的指令，然后被编译成不同的电机输出模式。揭示蜕皮症中神经编译的机制可能会深刻地帮助我们理解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。 为了研究这些问题，我们的实验室试图阐明控制果蝇幼虫和成虫蜕皮序列的回路，尽管在过去的一年里，我们主要关注幼虫蜕皮的神经元决定因素。如上所述，幼虫蜕皮严格依赖于内在因素，这一事实从观察到外源外周释放的激素-蜕皮触发激素(ETH)应用于孤立的幼虫神经系统足以触发神经元的顺序激活，这些神经元表达其他控制蜕皮的关键神经调节剂。证明这种顺序激活与广义假想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。为了识别这些目标，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木马外显子技术(Diao等人，2015年，细胞报告10,14101421)。特洛伊木马外显子能够识别和遗传操纵表达特定目的基因的神经元，我们使用这一工具选择性地靶向表达ETH受体/基因的两种异构体ETHRA和ETHRB的神经元。 正如今年发表的一篇论文(Diao等人，2016，Genetics 202：175-189)所描述的那样，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，并且在所有发育阶段都需要表达ETHRA而不是ETHRB的神经元。我们的遗传和神经元操作的结果表明，ETHRB在幼虫蜕皮和幼虫蜕皮中发挥重要作用，但不是幼虫蜕皮，这进一步证实了在幼虫蜕皮时，共表达激素甲壳类心脏活性多肽(CCAP)的ETHRA表达神经元的需求。在这项工作中，我们还鉴定了其他功能上重要的ETHRA表达神经元亚群，包括共同表达亮氨酸激动素的一个亚群。我们证明了Leucokinin表达的神经元在蛹阶段调节液体平衡以促进蜕皮。 因此，我们最近的工作增强了我们对神经元的理解，这些神经元作用于ETH下游，介导了蛹的蜕皮序列。如上所述，这些神经元包括表达其他激素的细胞，这些激素对产生幼虫蜕皮序列至关重要，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。这项工作还依赖于特洛伊木马外显子方法，我们成功地应用这一技术来阐明幼虫蜕皮电路，说明了绘制激素调节网络图的一般策略。使用受体基因作为遗传和神经元操作的切入点，可以在网络中不同级别的神经元之间建立功能连接的模式。 总而言之，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，并补充了其他关于果蝇神经系统功能结构的研究。正如去年发表的一篇特邀观点(White，B.H.，2016，J Neurogenet：30：54-61)所述，对果蝇和其他遗传模型动物神经系统组织的深入研究有望全面且迄今前所未有地理解神经系统如何在一生中仲裁动物的需求和欲望，并应为所有神经系统的一般运作提供不可或缺的见解。因此，这些洞察力应该有助于揭示行为组织中的缺陷，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.