Identifying Neural Substrates of Behavior in Drosophila Melanogaster

蜕皮序列是固定的运动程序，用于在每次蜕皮时脱掉昆虫的外骨骼，并扩展新的外骨骼以适应进一步的生长。因此，生存和生长取决于蜕皮序列的成功执行，而每一个蜕皮序列都必须根据动物的特定发育阶段进行调整。在我的实验室里，大部分研究的焦点都集中在新变形的成虫的发育阶段，在这个阶段，蜕化序列包括一个适应性的、对环境敏感的行为程序，这个程序介导了对合适环境的寻找，以及一个随后的、激素驱动的程序，这个程序用于扩大新出现的苍蝇最近发育的翅膀。正如今年发表在《昆虫学年度评论》（White & Ewer, 2014, Annu）上的一篇特邀评论所总结的那样。昆虫学报，59:363-381)，第一个程序是用来找到一个安全的栖息之处，在那里一动不动的苍蝇可以展开翅膀，第二个程序开始扩张。因为翅膀的扩张必须在出现的几个小时内进行，所以安全的需要必须与扩张的必要性相平衡，每只苍蝇必须决定何时（以及在什么样的环境条件下）扩张。因此，翅膀扩张为研究决策提供了一个行为范式，这是行为整合的最基本方面，也为理解激素和环境因素如何单独或协同作用，以招募运动模式来组装行为序列提供了一个行为范式。这样的理解，反过来，应该阐明行为组织的缺陷，这是许多精神障碍的根源，包括强迫症、精神分裂症和双相情感障碍。

Ecdysis sequences are stereotyped motor programs that are used to cast off an insects exoskeleton at each molt and to expand a new exoskeleton to accommodate further growth. Survival and growth thus depend upon successful execution of ecdysis sequences, and each such sequence must be tailored to the animals particular developmental stage. The developmental stage that has been the focus of most of the research in my laboratory is that of the newly metamorphosed adult, where the ecdysis sequence includes an adaptive, environmentally-sensitive behavioral program that mediates the search for suitable surroundings, and a subsequent, hormonally-driven program that serves to expand the recently developed wings of the newly emerged fly. As summarized in an invited review published this year in the Annual Reviews of Entomology (White & Ewer, 2014, Annu. Rev. Entomol. 59: 363-381), the first program is used to find a safe perch from which the immobile fly can expand its wings, and the second program initiates expansion. Because wing expansion must be undertaken within several hours of emergence, the need for safety must be balanced by the imperative to expand and each individual fly must decide when (and under what environmental circumstances) to expand. Wing expansion thus provides a behavioral paradigm for studying decision-making, the most fundamental aspect of behavioral integration, and for understanding how hormonal and environmental factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Such understanding should, in turn, shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. My laboratory's approach to understanding how the Drosophila nervous system produces behavioral sequences crucially depends on genetic techniques that allow specific subsets of brain cells to be turned off or on in freely behaving animals. This approach requires genetic tools for both suppressing and stimulating brain cell activity, as well as tools to target these manipulations to the desired subset of cells. Making such tools is another principal goal of our research. Two tools that have driven much of our recent research include a technique for acute neuronal activation using the mammalian cold-sensitive channel TRPM8, which we introduced in 2009 (Peabody et al., 2009, J Neurosci. 29:3343-53), and a technique for targeting transgene expression to neurons that express a specific gene of interest, which we introduced in 2012 (Diao & White, 2012, Genetics 190:1139-44). The ability to acutely activate neurons using TRPM8 was central to a project carried out in collaboration with the laboratory of Dr. Moto Yoshihara in which the goal was to develop a general method for identifying core circuit components responsible for generating simple behaviors in the fly. A manuscript describing this method was published during the reporting period in the journal G3 (Flood et al., 2013, G3:Genes, Genomes, Genetics 3:1629-37, and one of the principal successes of applying the approach was described in an earlier publication in Nature, which characterized a pair of command neurons that controls feeding behavior in Drosophila (Flood et al. , 2013, Nature 499:83-7). We have also used acute activation by TRPM8 to characterize the command neurons for wing expansion behavior, which we identified previously (Luan et al., 2012, J Neurosci 32: 880889). The recent work, published in the Journal of Experimental Biology (Peabody & White, 2013, J. Exp. Biol. 216:4395-4402), shows that the command neurons become competent to drive the wing expansion program only after emergence from the pupal case. Prior to this time the wing expansion network is suppressed--likely downstream of the command neurons--and the act of emergence, or some process closely associated with it, lifts this suppression. Our results provide insight into how nervous systems use inhibitory mechanisms to assemble motor programs into correctly ordered behavioral sequences. The T2A-Gal4 in-frame fusion technique couples the expression of transgenes to the expression of endogenous genes of interest using the ribosomal skipping mechanism of the viral T2A peptide. We had previously used this technique to make a fly line that co-expresses the Gal4 transcription factor in cells that express the receptor for bursicon, the hormone that governs wing expansion. In addition to using this line to characterize the functional roles of bursicon receptor-expressing neurons in the wing expansion, we also identified a novel set of cells in the fly gut that use bursicon signaling to regulate stem cell turnover. This work was carried out in collaboration with the laboratory of Dr. Marcos Vidal and was recently published in the journal Current Biology (Scopelliti et al., 2014, Curr. Biol. 24: 1199-1211). A second manuscript, in which the T2A technique was exploited to characterize the circuitry underlying color processing in the Drosophila brain, was the product of a collaboration with the laboratory of Dr. Chi-hon Lee and was published in the journal Neuron. (Karuppudurai et al., 2014, Neuron 81: 603-615). In addition to exploiting the T2A-Gal4 in-frame fusion technique to investigate specific biological questions, we have also spent considerable effort over the last two years further developing this technique. To extend its range of application, we have developed a broad "toolkit" of plasmids and fly lines that permits the technology to be easily used in conjunction with the MiMIC transposon-containing lines produced by the Drosophila Gene Disruption Project. Our "MiMIC-T2A-mediated In-Frame Fusion" method (i.e. MiMIC-TIFF) allows Gal4 and numerous other transgenes to be expressed in the same pattern as endogenous genes that contain MiMIC insertions. The method works for any MiMIC insertion that lies within the coding intron of a gene of interest and uses recombinase-mediated cassette exchange to replace the MiMIC insert with a T2A fusion construct preceded by a splice acceptor site. Currently, there are several thousand suitable MiMIC insertions available for use with this technique, and we have successfully tested the technique with T2A constructs that permit expression of not only Gal4, but also Gal80 and components of the Split Gal4 system, which we introduced in 2006 (Luan et al., 2006, Neuron, 52:425-36). A manuscript describing this technique is expected to be submitted later this year. A second manuscript in preparation describes the application of the MiMIC-TIFF technique to characterize neurons that express the receptor of Ecdysis Triggering Hormone, which is the master regulator of ecdysis sequences in flies and other insects. In summary, we have made substantial progress during the last year in elucidating, or helping to elucidate, several behavioral circuits in the Drosophila nervous system, including the one that supports wing expansion. At the same time, we have continued to develop tools that will 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 wing expansion circuit andmore broadly, the circuit that underlies ecdysis sequences at all developmental stagesour work should provide insight into the principles used by all nervous systems to generate and organize behavior