Nociceptive interneurons control modular motor pathways to promote escape behavior in Drosophila
- Anita Burgos
- Ken Honjo
- Tomoko Ohyama
- Cheng Sam Qian
- Grace Ji-eun Shin
- Daryl M Gohl
- Marion Silies
- W Daniel Tracey
- Marta Zlatic
- Albert Cardona
- Wesley B Grueber
- Columbia University Medical Center, United States
- University of Tsukuba, Japan
- McGill University, Canada
- University of Minnesota Genomics Center, United States
- European Neuroscience Institute Göttingen, Germany
- Indiana University, United States
- Howard Hughes Medical Institute, United States
- Columbia University, United States
Figures
Figure 1 with 4 supplements
Identification of candidate nociceptive interneurons.
(A) Schematic showing the Drosophila larval CNS. Red scaffold represents class IV (cIV) projections. Enlarged transverse section through ventral nerve cord (VNC) is shown below. Color-coded regions …
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Figure 1—source data 1
Summary table of graph data and statistical testing for thermogenetic activation experiments.
Genotypes, number of animals tested, graph data and statistical testing presented for thermogenetic activation experiments.
- https://doi.org/10.7554/eLife.26016.007
Figure 1—figure supplement 1
4051-Gal4 expression pattern.
(A) Expression pattern of 4051-Gal4. (B–B'') Co-labeling of 4051-Gal4 and 412-QF in DnB neurons (arrowheads). Some additional expression of 412-QF appeared in trachea after InSITE swapping …
Figure 1—figure supplement 2
Further analysis of 412-Gal4 expression.
(A–C') 412-Gal4, UAS-mCD8:GFP does not label cIV cell bodies or dendrites labeled by ppk-CD4-tdTomato (anti-GFP, green; anti-dsRed, red). (D) Expression of 412-Gal4 in single hemisegment. Two …
Figure 1—figure supplement 3
DnB polarity analysis.
(A–A') 412-Gal4 driven dendritic marker, DenMark (anti-dsRed, red) localizes to medial-directed projection, and medial arbors (arrow). UAS-mCD8:GFP (anti-GFP, green) is abundant throughout all …
Figure 1—figure supplement 4
412-Gal4, 4051-Gal4, and off target activation.
(A) Schematic representation of nocifensive escape behavior, which includes C-shaped body bending and 360˚ rolls. (B) Percentage of animals exhibiting rolling behavior during dTrpA1 activation …
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Figure 1—figure supplement 4—source data 1
Summary table of graph data and statistical testing for activation experiments.
Genotypes, number of animals tested, graph data and statistical testing presented for 412-Gal4, 4051-Gal4 and off-target activation experiments.
- https://doi.org/10.7554/eLife.26016.008
Figure 2
DnBs promote bending and rolling in a dose-dependent manner.
(A, C) Behavior ethograms upon optogenetic stimulation of 412-Gal4 or class IV neurons. Groups of animals expressing ReaChR in either population were subjected to optogenetic activation at different …
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Figure 2—source data 1
Summary table of behavioral responses to dose-dependent optogenetic activation.
Number of animals displaying bending, rolling, pausing, or crawling during optogenetic activation of ppk1.9-Gal4 or 412-Gal4 neurons across different levels of activation.
- https://doi.org/10.7554/eLife.26016.012
Figure 3
DnBs are activated by noxious heat downstream cIV sensory neurons.
(A) Representative heat maps showing Ca2+ responses in DnB cell bodies (arrowhead) before (~24˚C) and during (~44˚C) local noxious heat stimulation of the body wall. (B) Individual Ca2+ responses …
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Figure 3—source data 1
Summary table of graph data and statistical testing for functional imaging and nociceptive experiments.Genotypes, number of animals tested, graph data and statistical testing presented for DnB GCaMP imaging and nociceptive behavior experiments.
- https://doi.org/10.7554/eLife.26016.014
Figure 4 with 1 supplement
DnBs are required for body bending during nocifensive rolling.
(A) Labeling DnB neurons using R70F01-LexA driving 8X-Aop2-FLPL, and 412-Gal4 driving 10XUAS > Stop > Kir2.1-GFP (anti-GFP, green). (B) Global heat stimulus leads to rolling (top), transition period …
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Figure 4—source data 1
Summary table of graph data and statistical testing for silencing experiments.Genotypes, number of animals tested, graph data and statistical testing presented for R70F01∩412 silencing experiments and curvature analysis.
- https://doi.org/10.7554/eLife.26016.018
Figure 4—figure supplement 1
Intersectional labeling strategy, effect of silencing A27j neurons and effect of silencing 412-Gal4 neurons on somatosensory behavior.
(A) R70F01-LexA driven 13XLexAop2-IVS-myr::GFP labels DnBs and additional VNC and brain neurons (anti-GFP, green). (B–B') Chart showing distribution of expression patterns using R70F01∩412 to drive …
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Figure 4—figure supplement 1—source data 1
Summary table of graph data and statistical testing for silencing experiments.Genotypes, number of animals tested, graph data and statistical testing presented for silencing experiments on nociceptive, gentle-touch and crawling assays.
- https://doi.org/10.7554/eLife.26016.019
Figure 5 with 1 supplement
Connectome of sensory and interneuron inputs to DnB neurons.
(A) First instar larval brain with bilateral reconstruction of DnB neuron morphology in segment A1. Cyan and red dots indicate input and output synapses, respectively. Top, dorsal view; bottom, …
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Figure 5—source data 1
- https://doi.org/10.7554/eLife.26016.023
Figure 5—figure supplement 1
Down-and-Back neurons receive spatially segregated sensory input.
(A) Connectome of sensory input synapse onto DnB axon vs. dendrite in right and left A1 hemisegments. Numbers of synaptic connections between sensory neurons in top row and DnB neurons are shown. …
Figure 6 with 1 supplement
Connectome of DnB to premotor and nociceptive interneuron outputs.
(A) First instar larval CNS showing reconstruction of DnB neurons (green), and nociceptive integrating interneurons (purple). Output synapses are indicated in red and input synapses in cyan. …
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Figure 6—source data 1
Summary table for output connectivity graph.Percentage of top hit neurons (>3 synapses with DnB) that fall into the category: premotor, motor, or nociceptive integrator neurons.
- https://doi.org/10.7554/eLife.26016.026
Figure 6—figure supplement 1
Additional properties of Down-and-Back output signaling.
(A) mCD8:GFP driven by 412-Gal4, labeled with anti-GFP (green). (A') Cha3.3kb-Gal80 reduces expression of mCD8:GFP (anti-GFP, green) in DnB neurons. (B) DnB neuron (anti-GFP, green); (C') Boxed …
Figure 7 with 1 supplement
DnBs promote rolling, but not C-bending, through Goro network.
(A) Wiring diagram of DnB to Goro rolling command-like neuron. Percentage represents fraction of total dendritic inputs provided by upstream neuron class. Percentages may underestimate contribution …
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Figure 7—source data 1
Summary table of graph data and statistical testing for Goro functional imaging and behavior experiments.
Genotypes and maximum ∆F/F0 per animal tested during Goro imaging experiments. Genotypes, number of animals tested, graph data and statistical testing presented for 412-Gal4+Goro-experiments.
- https://doi.org/10.7554/eLife.26016.029
Figure 7—figure supplement 1
Silencing PMSI premotor neurons reduces rolling.
(A) Expression pattern of Per-Gal4, which includes PMSI premotor neurons. (B) Number of rolls decreases upon Per-Gal4 silencing with Kir2.1. (C) Percent of time spent engaged in Bend-crawl, Rolling, …
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Figure 7—figure supplement 1—source data 1
Summary table of graph data and statistical testing for PMSI silencing experiments.Genotypes, number of animals tested, graph data and statistical testing presented for PMSI silencing experiments.
- https://doi.org/10.7554/eLife.26016.030
Figure 8
Summary model for DnB neurons controlling nocifensive escape.
DnB neurons receive dual mechanosensory and nociceptive input, and promote nocifensive escape behavior via co-activation of downstream premotor circuits and command-like Goro neurons.
Videos
Video 1
412-Gal4 activation induces nocifensive rolling
Video shows result of activating 412-Gal4 neurons with dTrpA1 or ReaChR. For ReaChR videos, flashing light indicates ‘lights on.’
Video 2
Activating 412-Gal4 neurons in the VNC causes body bending
Video shows result of activating 412-Gal4 neurons exclusively in the brain or the VNC, compared to cIV activation
Video 3
DnB neurons are activated by noxious thermal stimuli
Video shows GCamp6m fluorescence in the VNC of a partially dissected larvae, with and without class IV neural activity. Arrows used to indicate cell body, axon, and dendrites.
Video 4
Silencing R70F01∩412 neurons reduces body curvature during rolling
Video shows defective rolling upon R70F01∩412 neuron silencing
Video 5
Activating 412-Gal4 while silencing Goro neurons biases larvae towards bending without rolling.
Video shows bending without rolling when 412-Gal4 neurons, including DnBs, are activated while suppressing Goro activity
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (D. melanogaster) | PB[IT.Gal4]0412 | PMID:21473015 | ||
| strain, strain background (D. melanogaster) | R70F01-LexA | PMID: 23063364 | RRID:BDSC_53628 | |
| strain, strain background (D. melanogaster) | R69E06-LexA | PMID: 23063364 | RRID:BDSC_54925 | |
| strain, strain background (D. melanogaster) | ppk1.9-Gal4 | PMID: 12956960 | ||
| strain, strain background (D. melanogaster) | 20X-UAS-IVS-GCaMP6m | PMID: 23868258 | RRID:BDSC_42748 | |
| strain, strain background (D. melanogaster) | UAS-dTrpA1 | PMID: 18548007 | RRID:BDSC_26263; RRID:BDSC_26264 | |
| strain, strain background (D. melanogaster) | UAS-ReaChR | PMID: 23995068 | RRID:BDSC_53749;RRID:BDSC_53741 | |
| strain, strain background (D. melanogaster) | tub > Gal80>; tsh-LexA, 8X-LexAop2-FLPL/CyO-RFP-tb; UAS-10X-IVS-myr:GFP | Gift from Dr. Marta Zlatic | ||
| strain, strain background (D. melanogaster) | tub > Gal80>; tsh-LexA, 8X-LexAop2-FLPL/CyO-RFP-tb; UAS-dTrpA1/TM6B | Gift from Dr. Marta Zlatic | ||
| strain, strain background (D. melanogaster) | UAS-TNT | PMID: 7857643 | RRID:BDSC_28838 | |
| strain, strain background (D. melanogaster) | UAS-TNTi | PMID: 7857643 | RRID:BDSC_28840 | |
| strain, strain background (D. melanogaster) | tsh-Gal80 | Gift from Dr. Julie Simpson | ||
| strain, strain background (D. melanogaster) | 8X-LexAop2FLPL; 10X-UAS > Stop > myr:GFP | PMID: 24183665 | ||
| strain, strain background (D. melanogaster) | 8X-LexAop2FLPL; 10X-UAS > Stop > Kir2.1-GFP | PMID: 24183665 | ||
| strain, strain background (D. melanogaster) | 13X-LexAop2-IVS-TNT::HA | PMID: 24507194 | Gift from Dr. Chi-Hon Lee | |
| strain, strain background (D. melanogaster) | LexAop-Kir2.1 | PMID: 24991958 | Gift from Dr. Barry Dickson | |
| strain, strain background (D. melanogaster) | 20xUAS-CsChrimson-mCherry | PMID: 27720450 | ||
| strain, strain background (D. melanogaster) | 13xLexAop2-IVS-GCaMP6s | PMID: 23868258 | ||
| strain, strain background (D. melanogaster) | yw; Mi{PTGFSTF.0} ChATMI04508-GFSTF.0 | PMID: 26102525 | ID_BSC: 60288 | |
| antibody | anti-GFP | Abcam | RRID: AB_300798 | 1:1000 |
| antibody | anti-DsRed | Clontech | RRID:AB_10013483 | 1:250 |
| antibody | anti-Fasciclin II | DSHB | RRID:AB_528235 | 1:100 |
| antibody | anti-5HT | Sigma | RRID:AB_477522 | 1:1000 |
| antibody | anti-dvGLUT | PMID: 15548661 | RRID:AB_2314347 | 1:10,000 |
| antibody | anti-GABA | Sigma | RRID:AB_477652 | 1:100 |
| antibody | anti-ChAT | DSHB | RRID:AB_2314170 | 1:100 |
Additional files
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Source code 1
Larval body curvature analysis.
- https://doi.org/10.7554/eLife.26016.033
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Source code 2
Generate kymograph from curvature analysis.
- https://doi.org/10.7554/eLife.26016.034
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Supplementary file 1
Main Figure genotypes.
- https://doi.org/10.7554/eLife.26016.035
