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A rapid and bidirectional reporter of neural activity reveals neural correlates of social behaviors in Drosophila

Nature Neuroscience volume 26, pages 1295–1307 (2023)Cite this article

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Abstract

Neural activity is modulated over different timescales encompassing subseconds to hours, reflecting changes in external environment, internal state and behavior. Using Drosophila as a model, we developed a rapid and bidirectional reporter that provides a cellular readout of recent neural activity. This reporter uses nuclear versus cytoplasmic distribution of CREB-regulated transcriptional co-activator (CRTC). Subcellular distribution of GFP-tagged CRTC (CRTC::GFP) bidirectionally changes on the order of minutes and reflects both increases and decreases in neural activity. We established an automated machine-learning-based routine for efficient quantification of reporter signal. Using this reporter, we demonstrate mating-evoked activation and inactivation of modulatory neurons. We further investigated the functional role of the master courtship regulator gene fruitless (fru) and show that fru is necessary to ensure activation of male arousal neurons by female cues. Together, our results establish CRTC::GFP as a bidirectional reporter of recent neural activity suitable for examining neural correlates in behavioral contexts.

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Fig. 1: Drosophila CRTC translocates into the nucleus upon odor-evoked neural activation.
Fig. 2: Subcellular distribution of CRTC::GFP provides a readout for neural activity in free-moving flies.
Fig. 3: Subcellular distribution of CRTC::GFP changes on the order of minutes and indicates both increases and decreases in neural activity.
Fig. 4: Automating pixel classification by deep convolutional neural network.
Fig. 5: CRTC::GFP detects acute increases and decreases in neural activity upon mating.
Fig. 6: Examination of the activity of P1a neurons in fru mutants.
Fig. 7: Examination of the activity of P1a neurons in food-deprived flies.

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Data availability

All numerical data are available in Supplementary Table 4. Information and requests for large raw data files, such as images and videos, should be directed to, and will be fulfilled by, D.H. Information and requests for resources and reagents should also be directed to, and will be fulfilled by, D.H.

Code availability

Data analysis codes, including neural network, are available for download from GitHub (https://github.com/hattorilabutsw/crtctoolkit/releases/) and are in Supplementary File 1.

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Acknowledgements

We thank W. Wojtowicz, H. Kramer and L. Zipursky for their comments on the manuscript; C. Rogine and R. Larios for technical assistance; K. Asahina, Y. Aso, G. Rubin, Z. Shang, M. Sieber, D. Smith and members of the Axel and Hattori laboratories for discussions; Y. Aso for sharing fly stock before publication; D. Anderson, K. Asahina, W. Grueber, M. Montminy, G. Struhl, Bloomington Drosophila Stock Center and Vienna Drosophila Resource Center for fly stocks; UT Southwestern Bioinformatics Core Facility for help with machine learning; and M. Alayon, R. Doris, C. Eccard, B. Fields, M. Gutierrez, P. Kisloff, A. Nemes and L. Taylor for administrative assistance. R.A. is a Howard Hughes Investigator. This work is supported by National Institutes of Health grant R01 DK132705, the Texas Rising STAR Award and the UT Southwestern Effie Marie Cain Scholarship to D.H.

Author information

Author notes

  1. These authors contributed equally: Moise Bonheur, Kurtis J. Swartz, Melissa G. Metcalf, Xinke Wen.

Authors and Affiliations

  1. Department of Physiology, UT Southwestern Medical Center, Dallas, TX, USA

    Moise Bonheur, Xinke Wen, Avirut Mehta, Kristin E. Connors & Daisuke Hattori

  2. Department of Neuroscience, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, USA

    Kurtis J. Swartz, Melissa G. Metcalf, Anna Zhukovskaya, Julia G. Barasch & Richard Axel

  3. Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, USA

    Andrew R. Jamieson

  4. Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA

    Kelsey C. Martin

  5. Simons Foundation, New York, NY, USA

    Kelsey C. Martin

  6. Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX, USA

    Daisuke Hattori

  7. Peter O’Donnell Jr. Brain Institute, UT Southwestern Medical Center, Dallas, TX, USA

    Daisuke Hattori

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Contributions

K.C.M., R.A. and D.H. conceived the project. M.B., K.J.S., M.G.M., X.W., A.Z., A.M., K.E.C., J.G.B. and D.H. performed experiments and analyzed data. A.R.J. and D.H. performed deep convolutional neural network training. R.A. and D.H. supervised the project. D.H. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Daisuke Hattori.

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Nature Neuroscience thanks Minoru Saitoe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Translocation of CRTC::GFP in DC3 PNs.

(a) ΔF/F0 traces from GCaMP calcium imaging experiments of DC3 projection neurons (PNs). Stimulus period is indicated by black line. Sample number represents the number of cell bodies imaged and number of flies in parenthesis. Lines indicate mean and shadings indicate SEM. (b) Changes in CRTC::GFP nuclear signals (NLI) in DC3 PNs in different conditions for Fig. 1d. Thin lines represent individual flies (averaged across cells) and thick lines indicate mean ± SEM across flies. (c) and (d) Changes in CRTC::GFP nuclear signals (NLI) in DC3 PNs in different conditions for Fig. 1e,h. Thin lines represent individual flies (averaged across cells) and thick lines indicate mean ± SEM across flies. ΔNLI in Fig. 1e,h was calculated for each cell by subtracting baseline NLI and averaged across cells per fly. (e) Estimated fraction of CRTC::GFP molecules in the nucleus for different NLIs in cells with different relative nuclear radii. The range of relative nuclear radii was determined to be 0.55 to 0.85 based on the cell and nuclear ROIs determined for 10,338 neurons of different cell types that were immunostained and imaged using confocal microscopy (0.71 ± 0.09; mean ± SD). For DC3 live imaging, mean relative nuclear radius is calculated based on images obtained using 2-photon microscope (0.70 ± 0.06; mean ± SD).

Extended Data Fig. 2 Subcellular distribution of CRTC::GFP provides a readout for neural activity in free moving flies.

(a) NLI of CRTC::GFP signal in PAM-γ4 for thermogenetic activation experiments. Individual fly data for Fig. 2b. Lighter color dots are individual cells and darker color dots are mean of each fly. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). (b) Confocal images of brains immunostained with anti-calcineurin-β antibody (see Methods). Maximum intensity projection images of slices that encompass antennal lobe to mushroom body lobes are shown. A pan-neuronal driver (c155, elav) was used to express shRNA targeting GFP (control, left) or CanB/B2 (right). Immunostaining for both control and experimental brains was performed in the same wells and the same imaging parameters were used for all brains. Representative images from 3 experiments (n = 12 for GFP, n = 12 for CanB/B2) are shown. (c) NLI of CRTC::GFP signal in the MBON-α‘3 (labeled by VT037580-GAL4) in the presence of RNAi-mediated knockdown of calcineurin B and B2 (CanB/B2). Each dot represents mean NLI across cells per fly. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). Statistics by two-sided Wilcoxon rank sum test; ***P < 0.001. (d) NLI of CRTC::GFP signal in MBON-α‘3 for thermogenetic activation experiments. Individual fly data for Fig. 2d. Lighter color dots are individual cells and darker color dots are mean of each fly. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). (e) Thermogenetic activation experiments for MBON-α‘3 using GFP instead of CRTC::GFP. Each dot represents mean NLI across cells per fly. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). Kruskal-Wallis test followed by a post hoc Tukey’s HSD test; P = 0.002 for comparisons between dTRPA1+, 32 °C and dTRPA1-, 25 °C. (f) NLI of CRTC::GFP signal in P1a neurons of male flies in different rearing conditions. Individual fly data for Fig. 2f. Lighter color dots are individual cells and darker color dots are mean of each fly. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). (g) NLI of CRTC::GFP signal in P1a neurons of male flies in different rearing conditions. Subject flies were housed singly for 2 days upon eclosion and were housed for 1 day with males or females, or were left single-housed (‘solo’). Each dot represents mean NLI across cells per fly. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). Statistics by Kruskal-Wallis test followed by a post hoc Tukey’s HSD test; *P < 0.05.

Extended Data Fig. 3 Evaluation of network performance.

(a) Performance evaluation for trained U-net, MobileNet-v2, ResNet-18, and Xception. (b) Eleven examples of pixel classification by trained ResNet-50 (UbwonkoNet) and trained U-net. The cells in the 4th and 8th rows show examples where U-Net failed to selectively ‘focus’ on the cell body of interest. Examples include those shown in Fig. 4c. (c) Comparison of experimental results obtained using UbwonkoNet-determined NLIs vs manually-determined NLIs. A subset of data from the P1a experiments described in Fig. 7 (ad libitum fed group) were manually quantified (purple) and compared to the results obtained by UbwonkoNet (green). The left two columns are the same as the first and the third column of Fig. 7b. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). Statistics by Kruskal-Wallis test followed by a post hoc Tukey’s HSD test; ***P < 0.001.

Extended Data Fig. 4 Neuronal expression of CRTC::GFP does not significantly affect male mating and courtship behaviors.

(a) Fertility of flies expressing GFP, CRTC::GFP, or EGFP::Kir2.1 in corazonin neurons. n = 25 each. Statistics by Fisher’s exact test and Bonferroni correction; ***P < 0.001. (b) Behavior observation chamber used to examine mating behaviors. (c) Left; ethograms of mating experiments. Males of indicated genotypes were paired with wild-type females in the behavior observation chamber. Middle; copulation duration in 2-hour observation period for fly pairs each consisting of a male of indicated genotype and a wild-type female. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). Statistics by Kruskal-Wallis test followed by a post hoc Tukey’s HSD test; ***P < 0.001. Right; latency to copulation initiation. Statistics by Kruskal-Wallis test followed by a post hoc Tukey’s HSD test; P > 0.05. (d) Courtship and mating behaviors of male flies expressing CRTC::GFP pan-neuronally. Left; ethograms. Right; courtship index. Boxplot; median (horizontal line), first and third quartiles (box), and data within 1.5x IQR (whiskers). Statistics by two-sided Wilcoxon rank sum test; P > 0.05. (e) Behavioral transitions during courtship behavior of male flies expressing CRTC::GFP pan-neuronally. Behaviors aligned to the onset (left) or offset (right) of licking (top) or attempted copulation (bottom). Line plots show fraction of epochs with specific behavior over time (thick lines) and shading indicates 95% confidence interval determined by bootstrapping. n = 132 epochs for licking and n = 112 epochs for attempted copulation for elav>GFP, n = 167 and 132 for elav>CRTC::GFP.

Extended Data Fig. 5 Examination of P1a activity in fruitless mutant flies.

(a) Additional example flies for P1a calcium imaging. (b) P1a GCaMP response in contact epochs. Unsorted data for Fig. 6f. Calcium traces are shown only for the duration of each contact epoch, which varies from epoch to epoch. (c) Fraction of contact epochs in which P1a neurons become activated as determined with a different threshold from Fig. 6g. Activation epoch if at least 5 consecutive frames (appx. 0.28 sec) exhibit over 1.96 z-scored activity. Statistics by Fisher’s exact test. ***P < 0.001, n.s. P > 0.05.

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Bonheur, M., Swartz, K.J., Metcalf, M.G. et al. A rapid and bidirectional reporter of neural activity reveals neural correlates of social behaviors in Drosophila. Nat Neurosci 26, 1295–1307 (2023). https://doi.org/10.1038/s41593-023-01357-w

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