Whole-brain in situ mapping of neuronal activation in Drosophila during social behaviors and optogenetic stimulation
Abstract
Monitoring neuronal activity at single-cell resolution in freely moving Drosophila engaged in social behaviors is challenging because of their small size and lack of transparency. Extant methods, such as Flyception, are highly invasive. Whole-brain calcium imaging in head-fixed, walking flies is feasible but the animals cannot perform the consummatory phases of social behaviors like aggression or mating under these conditions. This has left open the fundamental question of whether neurons identified as functionally important for such behaviors using loss- or gain-of-function screens are actually active during the natural performance of such behaviors, and if so during which phase(s). Here, we perform brain-wide mapping of active cells expressing the Immediate Early Gene hr38 using a high-sensitivity/low background fluorescence in situ hybridization (FISH) amplification method called HCR-3.0. Using double-labeling for hr38 mRNA and for GFP, we describe the activity of several classes of aggression-promoting neurons during courtship and aggression, including P1a cells, an intensively studied population of male-specific interneurons. Using HI-FISH in combination with optogenetic activation of aggression-promoting neurons (opto-HI-FISH), we identify candidate downstream functional targets of these cells in a brain-wide, unbiased manner. Finally, we compare the activity of P1a neurons during sequential performance of courtship and aggression, using intronic vs. exonic hr38 probes to differentiate newly synthesized nuclear transcripts from cytoplasmic transcripts synthesized at an earlier time. These data provide evidence suggesting that different subsets of P1a neurons may be active during courtship vs. aggression. HI-FISH and associated methods may help to fill an important lacuna in the armamentarium of tools for neural circuit analysis in Drosophila.
Copyright and License
© 2023, Watanabe et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Acknowledgement
We thank NA Pierce and M Schwarzkopf for advice on HCR techniques; A Sanchez for fly stock maintenance; S Cao for advice on imaging experiments; B Weissbourd for advice on catFISH experiments; GM Rubin, BD Pfeiffer, and S Waddell for reagents; C Chiu, G Mancuso, and L Chavarria for the laboratory management and administrative assistance; and members of the Anderson laboratory for valuable comments on this work. This work was supported by NIDA Grant R01 DA031389. DJA is an Investigator of the Howard Hughes Medical Institute.
Data Availability
The data and code used for analyses in this paper are available in the GitHub repository (https://github.com/kiichiwatanabelab/Hr38_eLife, copy archived at Watanabe, 2024), and Dryad data repository (https://doi.org/10.5061/dryad.dr7sqvb7d). Any additional resources and reagents are available from the corresponding author upon request.
Watanabe et al. (2024) Dryad Digital Repository Data from: Whole brain in situ mapping of neuronal activation in Drosophila during social behaviors and optogenetic stimulation.
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Additional details
- National Institute on Drug Abuse
- DA031389
- Howard Hughes Medical Institute
- Available
-
2024-11-28Version of record online
- Caltech groups
- Division of Biology and Biological Engineering
- Publication Status
- Published