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 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 neural circuits can be variably configured to generate immensely 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 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 issues, 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. With regard to pupal ecdysis, we have been following up on our recently published description of the pupal ecdysis sequence at single muscle resolution (Elliott et al., eLife 2021;10:e68656). The motivation for that study, which to our knowledge constituted the first muscle-level description of a complicated behavioral sequence in any animal, was to facilitate a similar investigation of the neuronal activity that drives pupal ecdysis. This is in line with the laboratorys central interest in understanding 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. To study the patterns of neuronal activity that transform the ETH signal into a pupal ecdysis sequence, we plan to use an excised pupal brain preparation that we and others have previously shown exhibits fictive pupal ecdysis activity in response to ETH (Diao et al., 2017, doi.org/10.7554/eLife.29797). We plan to first establish that patterns of ETH-evoked motor neuron activity in the excised brain correlate well with the previously established patterns of pupal ecdysis muscle activity. We will then use the motor neuron activity patterns to identify the pre-motor neurons likely to drive them based on their patterns of activity. Using correlated activity and functional manipulations as our guide, we will identify neurons at progressively higher levels that participate in generating the movements of the pupal ecdysis sequence, up to and including the neurons that respond to ETH. Over the past year, we have made progress in implementing this strategy. We have characterized several so-called Gal4 driver lines that will allow us to reliably target either the general population of motor neurons, subsets of motor neurons, or subsets of premotor neurons. Preliminary studies of these drivers show considerable promise using conventional confocal microscopy. Because confocal microscopy lacks the resolution to allow 4D imaging of activity over the entire pupal brain in response to ETH, we have devoted much of our effort over the past year to completing installation of a custom-built light-sheet microscope. Installation was unfortunately seriously impacted by Covid-related restrictions, which delayed the configuration and set-up of servers designed to acquire, analyze, and store the imaging data. While optimization of server and microscope operation is on-going, we anticipate that we will be able to make rapid progress on pupal brain imaging in FY2023. A major focus of our study of the adult ecdysis circuit over the past year has continued to be the dual roles of the hormone Bursicon in wing expansion. Our previous work had shown that Bursicon released from a single pair of neurons (called the BSEG) was important in helping flies decide whether to expand their wings after adult ecdysis depending on environmental conditions. Expanding under adverse conditions risks permanently damaging the wings and flies will generally delay wing expansion and seek better conditions rather than risk damage. Using the Trojan exon technology that we developed in 2015 (Diao et al., 2015, Cell Rep. 10:1410-21) to investigate targets of Bursicon, we established that a key component of the decision-making circuitry is a set of cholinergic neurons that express the Bursicon receptor. These neurons signal back to the BSEG via a positive feedback loop to initiate and maintain wing expansion in an environmentally sensitive manner. This work was posted to the BioRxiv server and also submitted for publication. Based on comments on the posted manuscript and the reviews received, we have sought to further refine our identification of the cholinergic neurons that mediate feedback using both conventional methods and a novel split intein-based method discussed in the next paragraph. This work is on-going and is expected to be completed in FY2023. In technology development, my laboratory has continued to refine methods that exploit the use of split inteins. In a continuation of previous work, we are adapting split inteins for use in a modified version of the Split Gal4 method introduced by my laboratory in 2006 (Neuron 52:425-436). The modified version will have the advantage of being compatible with the temperature-sensitive Gal4 inhibitor tsGal80, and will therefore permit temporal control of transcriptional activity of the new Split Gal4 system. In addition, we are also using split inteins to modify our Trojan exon method. That method necessarily truncates the allele of the gene into which the Trojan exon is inserted, essentially cutting the encoded protein in half. To obviate this, we have devised a strategy in which split inteins are used to re-unite the two halves of the truncated protein thereby restoring its function. This method is proving useful in creating new Split Gal4 drivers to identify the cholinergic feedback neurons described above. More generally, it should prove to be a valuable tool to the Drosophila research community by extending the range of problems that can be tackled using the Trojan exon technique. 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 develop 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, our work should provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.