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 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, with our primary efforts over the last year being devoted to the circuitry governing 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. Evidence indicates that this sequential activation corresponds to the generation of a fictive ecdysis sequence, and our efforts over the last year have focused on characterizing the neuronal substrates of this fictive sequence using functional manipulations and Ca++ imaging. As we have previously shown, the major downstream targets of ETH include neurons that release the hormones Bursicon and CCAP (Diao et al., 2016, Genetics. 202:175-89. doi: 10.1534/genetics.115.182121). Both factors act within the nervous system as neuromodulators and have been shown to be important determinants of ecdysis sequences in all insects. However, little is known about the brain cells that mediate the effects of these factors on behavior. To identify these cells, we used the Trojan exon technique (Diao et al., 2015, Cell Rep. 10:1410-21. doi: 10.1016/j.celrep.2015.01.059) to target and functionally characterize neurons that express the receptors for Bursicon and CCAP. As reported in a paper published last year in Elife (Diao et al., 2017, Elife Nov 22;6. pii: e29797. doi: 10.7554/eLife.29797), we found that each hormone acts on a functionally distinct set of neurons within the network that generates the pupal ecdysis sequence: Bursicon targets essential components of a multifunctional central pattern generator that produces the three abdominal rhythms of the pupal ecdysis sequence, while CCAP targets the motor neurons that receive the output of the central pattern generator. The neuronal targets of these two hormones, together with the neurons targeted by the initiating signal, ETH, represent three hierarchically organized layers of the neural network governing ecdysis. Remarkably, the architecture of the pupal ecdysis network is thus revealed by mapping the sites of action of the key neuromodulators governing its behavioral output. This result suggests that the neuromodulatory determinants of a behavioral state act at all levels of the neural network regulating that state to bias and coordinate specific sensorimotor outcomes. During the past year, we have also contributed to collaborative research that resulted in papers published in Elife (Lee et al., 2018, Elife, Feb 23;7. pii: e33007. doi: 10.7554/eLife.33007) and Cell Reports (Xie et al., 2018, Cell Rep. 23(2):652-665. doi: 10.1016/j.celrep.2018.03.068.) Both publications exploit tools developed in our laboratory. The first paper describes the use of our Trojan exon technique (Diao et al., 2015, Cell Rep. 10:1410-21. doi: 10.1016/j.celrep.2015.01.059) to identify a novel protein kinase involved in long-term memory in Drosophila. The second paper describes a toolkit for targeting distinct populations of dopaminergic neurons in the fly brain. This toolkit includes fly lines that express the Killer Zipper, which we designed to augment the targeting capabilities of our Split Gal4 system (Dolan et al., 2017, Genetics 206:775-784. doi: 10.1534/genetics.116.199687 ). In summary, we have made good progress during the last year in elucidating the functional architecture of the neural network that underlies execution of the pupal ecdysis sequence. 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 at all developmental stages in the fly, our work should provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.
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