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，蛹蜕皮和成虫蜕皮在变态前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，然后编译成不同的输出。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，我的实验室试图阐明果蝇中控制蛹和成虫蜕皮序列的电路。在过去的一年里，我们的努力或多或少地平均分配在这两个电路的研究上。此外，我们已经投入了相当大的努力，以开发一种新的方法，遗传靶向小子集的神经元在苍蝇的大脑操纵。这种方法是我们实验室和其他实验室所需要的，以促进神经元回路的精细映射。2018年开展的三个项目中，每个项目都有很长的时间跨度，每个项目现在才开始撰写出版物。 关于蛹蜕皮，过去一年进行的工作偏离了我们对神经回路的典型关注，而是更仔细地描述了大脑产生的行为。这种焦点的转移源于这样一种认识，即如果我们要详细了解苍蝇大脑如何产生蛹蜕皮序列，我们必须首先详细了解这种行为的构成。为此目的，适当的描述水平是在蛹蜕皮过程中产生动物执行的运动的个体肌肉收缩。因此，我们已经开发了工具，全面监测整个蛹肌肉组织的活动在蜕皮。我们使用遗传编码的Ca++指标来生成肌肉活动的时间序列数据。这些数据根据所有个体肌肉收缩来定义蜕皮顺序。实现这一目标需要绘制整个蛹的肌肉组织及其神经支配模式。此外，为了证实肌肉收缩是由神经驱动的，我们开发了同时成像肌肉活动及其突触输入的方法。为了确定哪些输入受到甲壳动物生物活性肽（CCAP）的调节，如我们2017年eLIFE论文（Diao et al.，doi：10.7554/eLife.29797），我们已经鉴定了表达CCAP受体的运动神经元的补体。这项工作目前正在准备出版。 我们的成人蜕皮电路的研究集中在神经机制，调解环境敏感的决定，延迟翼扩张在不利条件下。我们以前的工作表明，这种决定可能是由分泌激素Bursicon的两个神经元（即BSEG）介导的（Luan等人，2012，J Neurosci. 32：880889）。使用我们在2015年开发的特洛伊外显子技术（Diao et al.，10：1410-21.土井：10.1016/j.celrep.2015.01.059），我们已经表征了BSEG的下游靶标，并且令人惊讶地发现，它们包括一组胆碱能神经元，其通过正反馈回路向BSEG发回信号。这个反馈回路对于启动和保持机翼展开是至关重要的，因此是决策电路的一个关键组成部分。这项工作也正在准备出版。 我们在过去一年中开发的新技术涉及使用分裂内含肽，这是最近发现的蛋白质，可以将两个蛋白质片段融合在一起。分裂内含肽本身自然地适用于蛋白质分子被分成惰性片段的应用，这些片段必须连接在一起才能重建活性。我们的方法将Cre重组酶分成三个片段，与两对不同的分裂内含肽融合。因此，这三种组分可以独立地靶向不同的细胞群，并且只有表达所有三种Cre组分的那些细胞才会产生活性Cre。Cre本身用于激活转录因子Gal 4，从而允许其他转基因在靶细胞中表达，以便可以操纵它们以确定它们的功能。描述这种方法的手稿目前正在准备提交，但我们已经与许多同事分享了我们未发表的试剂，以促进他们的电路映射工作。 总之，我们在过去的一年里在推进实验室感兴趣的主要问题的研究方面取得了良好的进展。与此同时，我们继续贡献工具，不仅支持我们自己的电路映射工作，也支持果蝇研究社区的其他成员。当我们使用这些工具来扩展和完善我们对蜕皮序列背后的电路的分析时，我们的工作应该提供对所有生物体（包括人类）中行为电路的发展和功能的原则的洞察。

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 ecdysisbefore and after metamorphosis, respectivelyare 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 outputs. 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, my laboratory seeks to elucidate the circuitry that governs both the pupal and adult ecdysis sequences in Drosophila. Our efforts over the last year have been more or less evenly divided between study of these two circuits. In addition, we have devoted considerable effort to developing a novel methodology for genetically targeting small subsets of neurons in the fly brain for manipulation. Such methods are required to facilitate the fine-mapping of neuronal circuits by our lab and others. Each of the three projects pursued in 2018 had a significant time horizon and each is only now being written up for publication. With regard to pupal ecdysis, the work conducted over the past year departs from our typical focus on neural circuitry and involves more carefully describing the behavior being generated by the brain. This shift in focus stemmed from the realization that if we are to understand in detail how the fly brain generates a pupal ecdysis sequence, we must first understand in detail what constitutes that behavior. The appropriate level of description for this purpose is that of the individual muscle contractions that produce the movements executed by the animal during performance of the pupal ecdysis sequence. Accordingly, we have developed tools to comprehensively monitor the activity of the entire pupal musculature during ecdysis. We use genetically-encoded Ca++ indicators to generate time-series data of muscle activity. These data define the ecdysis sequence in terms of all the individual muscle contractions. Achieving this objective has required mapping the entire pupal musculature and its pattern of innervation. In addition, to confirm that muscle contractions are neurally driven, we have developed methods for simultaneously imaging the activity of both muscles and their synaptic inputs. To determine which inputs are subject to modulation by Crustacean Cardioactive Peptide (CCAP), as described in our 2017 eLIFE paper (Diao et al., doi: 10.7554/eLife.29797), we have identified the complement of motor neurons that express the CCAP receptor. This work is currently being prepared for publication. Our study of the adult ecdysis circuit has focused on the neural mechanisms that mediate the environmentally-sensitive decision to delay wing expansion under adverse conditions. Our previous work had shown that this decision was likely mediated by two neurons (i.e. the BSEG) that secrete the hormone Bursicon (Luan et al., 2012, J Neurosci. 32: 880889). Using the Trojan exon technology that we developed in 2015 (Diao et al., 2015, Cell Rep. 10:1410-21. doi: 10.1016/j.celrep.2015.01.059), we have characterized downstream targets of the BSEG and found, surprisingly, that they include a set of cholinergic neurons that signal back to the BSEG via a positive feedback loop. This feedback loop is critical for initiating and maintaining wing expansion and thus represents a key component of the decision-making circuitry. This work is likewise being prepared for publication. The novel technology that we have developed during the past year involves the use of split inteins, which are recently discovered proteins that can join two protein fragments fused to them. Split inteins lend themselves naturally to applications in which a protein molecule is divided into inert fragments that must be joined together to reconstitute activity. Our method divides the Cre recombinase into three fragments fused to two distinct pairs of split inteins. The three components can thus be independently targeted to different cell groups and only those cells that express all three Cre components will generate active Cre. Cre itself is used to activate a transcription factor, Gal4, thus allowing expression of other transgenes in the targeted cells so that they can be manipulated to determine their function. A manuscript describing this methodology is currently being prepared for submission, but we have already shared our unpublished reagents with numerous colleagues to facilitate their circuit-mapping efforts. In summary, we have made good progress during the last year in advancing research on the principal questions of interest to the laboratory. At the same time, we have continued to contribute tools that support not only our own circuit mapping efforts, but also those of other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the circuitry underlying ecdysis sequences, our work should provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.