Identifying Neural Substrates of Behavior in Drosophila Melanogaster

National Institute Of Mental Health

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Abstract

Insect ecdysis sequences represent a simple, robust, and tractable model for studying the neuromodulatory mechanisms that govern behavioral state. In addition, because ecdysis behaviors are inherently sequential, they permit the systematic investigation of how motor programs are assembled for serial execution by the nervous system. Finally, the study of ecdysis sequences promises insight into how neural circuits can be variably configured to generate very different behaviors. In Drosophila, for example, the motor sequences performed at pupal and adult ecdysis are completely different. This is because of the profound differences in the pupal and adult body plans. Despite these anatomical differences, the two behavioral sequences are governed by a common set of neuromodulatory/hormonal factors. 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 neural mechanisms of “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 issues, my laboratory seeks to elucidate the networks that govern both the pupal and adult ecdysis sequences in Drosophila. In addition, we build genetic tools to facilitate the refined targeting of specific cell types in the fly—with a specific interest in using them to manipulate the function of neuromodulatory neurons. Our efforts over the last year have been more or less evenly divided between study of pupal and adult ecdysis and tool development. With regard to pupal ecdysis, we have built upon our previously published description of the pupal ecdysis sequence at single muscle resolution (Elliott et al., eLife 2021;10:e68656) and are now using lightsheet microscopy to similarly define the behavioral sequence at single motor neuron resolution. The motivation is to establish an experimental paradigm in which we can determine how a simple hormonal signal (i.e. ETH) is transformed in the brain into the execution of a specific motor sequence characterizing a particular behavioral state. This on-going work has been complemented by efforts to comprehensively characterize the motor programs underlying adult ecdysis using measurements of muscle Ca++ activity. Adult ecdysis immediately follows metamorphosis and consists of two major behavioral phases. In the first, the animal extricates itself from the puparium and in the second, it stops to expand and harden its wings. Each of these phases involves the execution of specific motor programs that are under hormonal control. We have previously characterized the behavioral mechanisms responsible for wing expansion. We are now characterizing the motor programs responsible for extrication, otherwise known as “eclosion.” Using the same strategy that we previously used to characterize pupal ecdysis behavior (Elliott et al., eLife 2021;10:e68656), we are imaging muscle activity through the puparium using the fluorescent calcium indicator GCaMP7s and are identifying what changes in muscle activity occur in response to manipulations of hormonal signaling. This is work in progress. In technology development, my laboratory has continued to generate driver lines useful to the community based on the various versions of our Split Gal4 and Trojan exon methods, described in earlier publications (Luan et al., 2006, Neuron 52:425-436; Diao et al., 2015, Cell Rep. 10:1410-21; Ewen-Campen et al., 2023, Proc Natl Acad Sci USA., 120 (24): e2304730120; Diao et al., 2024, Proc Natl Acad Sci USA. 121 (17): e2317083121). Of particular importance to our work on the ecdysis networks, we made critical Gal4 and Split Gal4 driver lines for a collaboration with the laboratory of Dr. John Ewer at the Universidad Valparaiso, Chile. This collaboration sought to identify and characterize the receptor for Eclosion Hormone (EH) in Drosophila. Together with ETH, EH is among the best-studied ecdysis hormones in insects. Despite this, however, its receptor in Drosophila and the cells that express it have remained enigmatic. In 2009, the gene encoding an EH receptor (EHR) was described in the distantly related oriental fruit fly, B. dorsalis, and to determine the function and expression pattern of the orthologous gene in Drosophila we made driver lines to permit the specific targeting of cells that expressed this gene. The manuscript describing this work is currently in press at PLoS Genetics. It demonstrates that the Drosophila EHR is expressed in many cell types, often together with the ETH receptor. This co-expression indicates that EH and ETH signaling includes not only previously described positive feedback interactions, but also convergent feed-forward pathways. This architecture serves to commit the animal to ecdysis, by stabilizing its behavioral state, while also driving the progressive execution of ecdysis behaviors. Such an architecture may prove to be a general feature of networks that regulate behavioral state. In summary, during the last year we have advanced research on the principal questions of interest to the laboratory. At the same time, we have continued to create technology that supports not only our own circuit mapping efforts, but also those of other researchers. As we use these tools to extend and refine our analysis of the circuitry underlying ecdysis sequences, we expect that our work will provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.