Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior

Abstract

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, we uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. We further show that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. Our findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: A08n neurons receive synaptic input from C4da neurons.
Figure 2: DP-ilp7 neurons are connected to multiple classes of sensory neurons.
Figure 3: A08n neurons are necessary and sufficient for nociceptive responses downstream of C4da neurons.
Figure 4: DP-ilp7 neurons integrate multi-mechanosensory input and facilitate nociceptive behavior.
Figure 5: sNPF signaling from DP-ilp7 to mechanosensory neurons is necessary for mechanonociceptive behavior.
Figure 6: Mechanonociceptive circuit elements and sNPF-R function are not required for C4da neuron-dependent thermonociception.
Figure 7: DP-ilp7 activity and sNPF signaling are required for C4da neuron presynaptic facilitation and A08n neuron responses.

References

  1. Dubin, A.E. & Patapoutian, A. Nociceptors: the sensors of the pain pathway. J. Clin. Invest. 120, 3760–3772 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Basbaum, A.I., Bautista, D.M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Lumpkin, E.A. & Caterina, M.J. Mechanisms of sensory transduction in the skin. Nature 445, 858–865 (2007).

    CAS  PubMed  Google Scholar 

  4. Taghert, P.H. & Nitabach, M.N. Peptide neuromodulation in invertebrate model systems. Neuron 76, 82–97 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Marder, E. Neuromodulation of neuronal circuits: back to the future. Neuron 76, 1–11 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Bargmann, C.I. Beyond the connectome: how neuromodulators shape neural circuits. BioEssays 34, 458–465 (2012).

    CAS  PubMed  Google Scholar 

  7. Nässel, D.R. & Winther, A.M.E. Drosophila neuropeptides in regulation of physiology and behavior. Prog. Neurobiol. 92, 42–104 (2010).

    PubMed  Google Scholar 

  8. Im, S.H. et al. Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila. Elife 4, e10735 (2015).

    PubMed  PubMed Central  Google Scholar 

  9. Ghysen, A., Dambly-Chaudière, C., Aceves, E., Jan, L.-Y. & Jan, Y.-N. Sensory neurons and peripheral pathways in Drosophila embryos. Rouxs Arch. Dev. Biol. 195, 281–289 (1986).

    PubMed  Google Scholar 

  10. Bodmer, R. & Jan, Y.N. Morphological differentiation of the embryonic peripheral neurons in Drosophila. Rouxs Arch. Dev. Biol. 196, 69–77 (1987).

    PubMed  Google Scholar 

  11. Grueber, W.B., Jan, L.Y. & Jan, Y.N. Tiling of the Drosophila epidermis by multidendritic sensory neurons. Development 129, 2867–2878 (2002).

    CAS  PubMed  Google Scholar 

  12. Tsubouchi, A., Caldwell, J.C. & Tracey, W.D. Dendritic filopodia, Ripped Pocket, NOMPC, and NMDARs contribute to the sense of touch in Drosophila larvae. Curr. Biol. 22, 2124–2134 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Yan, Z. et al. Drosophila NOMPC is a mechanotransduction channel subunit for gentle-touch sensation. Nature 493, 221–225 (2013).

    CAS  PubMed  Google Scholar 

  14. Tracey, W.D. Jr., Wilson, R.I., Laurent, G. & Benzer, S. painless, a Drosophila gene essential for nociception. Cell 113, 261–273 (2003).

    CAS  PubMed  Google Scholar 

  15. Zhong, L., Hwang, R.Y. & Tracey, W.D. Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae. Curr. Biol. 20, 429–434 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhong, L. et al. Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP channel. Cell Rep. 1, 43–55 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Gorczyca, D.A. et al. Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila. Cell Rep. 9, 1446–1458 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Guo, Y., Wang, Y., Wang, Q. & Wang, Z. The role of PPK26 in Drosophila larval mechanical nociception. Cell Rep. 9, 1183–1190 (2014).

    CAS  PubMed  Google Scholar 

  19. Kim, S.E., Coste, B., Chadha, A., Cook, B. & Patapoutian, A. The role of Drosophila Piezo in mechanical nociception. Nature 483, 209–212 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Neely, G.G. et al. A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene. Cell 143, 628–638 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Xiang, Y. et al. Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature 468, 921–926 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Hwang, R.Y. et al. Nociceptive neurons protect Drosophila larvae from parasitoid wasps. Curr. Biol. 17, 2105–2116 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Vogelstein, J.T. et al. Discovery of brainwide neural-behavioral maps via multiscale unsupervised structure learning. Science 344, 386–392 (2014).

    CAS  PubMed  Google Scholar 

  24. Ohyama, T. et al. A multilevel multimodal circuit enhances action selection in Drosophila. Nature 520, 633–639 (2015).

    CAS  PubMed  Google Scholar 

  25. Fetsch, C.R., DeAngelis, G.C. & Angelaki, D.E. Bridging the gap between theories of sensory cue integration and the physiology of multisensory neurons. Nat. Rev. Neurosci. 14, 429–442 (2013).

    CAS  PubMed  Google Scholar 

  26. van Atteveldt, N., Murray, M.M., Thut, G. & Schroeder, C.E. Multisensory integration: flexible use of general operations. Neuron 81, 1240–1253 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Stein, B.E., Stanford, T.R. & Rowland, B.A. Development of multisensory integration from the perspective of the individual neuron. Nat. Rev. Neurosci. 15, 520–535 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Gohl, D.M. et al. A versatile in vivo system for directed dissection of gene expression patterns. Nat. Methods 8, 231–237 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Frank, D.D., Jouandet, G.C., Kearney, P.J., Macpherson, L.J. & Gallio, M. Temperature representation in the Drosophila brain. Nature 519, 358–361 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Miguel-Aliaga, I., Thor, S. & Gould, A.P. Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons. PLoS Biol. 6, e58 (2008).

    PubMed  PubMed Central  Google Scholar 

  31. Grueber, W.B. et al. Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology. Development 134, 55–64 (2007).

    CAS  PubMed  Google Scholar 

  32. Klapoetke, N.C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Yang, L. et al. Trim9 regulates activity-dependent fine-scale topography in Drosophila. Curr. Biol. 24, 1024–1030 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang, W., Yan, Z., Li, B., Jan, L.Y. & Jan, Y.N. Identification of motor neurons and a mechanosensitive sensory neuron in the defecation circuitry of Drosophila larvae. Elife 3, e03293 (2014).

    PubMed Central  Google Scholar 

  35. Robertson, J.L., Tsubouchi, A. & Tracey, W.D. Larval defense against attack from parasitoid wasps requires nociceptive neurons. PLoS One 8, e78704 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Turner, H.N. et al. The TRP channels Pkd2, NompC, and Trpm act in cold-sensing neurons to mediate unique aversive behaviors to noxious cold in Drosophila. Curr. Biol. 26, 3116–3128 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Carlsson, M.A., Enell, L.E. & Nässel, D.R. Distribution of short neuropeptide F and its receptor in neuronal circuits related to feeding in larval Drosophila. Cell Tissue Res. 353, 511–523 (2013).

    CAS  PubMed  Google Scholar 

  38. Nässel, D.R., Enell, L.E., Santos, J.G., Wegener, C. & Johard, H.A.D. A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions. BMC Neurosci. 9, 90 (2008).

    PubMed  PubMed Central  Google Scholar 

  39. Lee, K.-S. Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling. Nat. Cell Biol. 10, 468–475 (2008).

    CAS  PubMed  Google Scholar 

  40. Abraira, V.E. & Ginty, D.D. The sensory neurons of touch. Neuron 79, 618–639 (2013).

    CAS  PubMed  Google Scholar 

  41. Seal, R.P. et al. Injury-induced mechanical hypersensitivity requires C-low threshold mechanoreceptors. Nature 462, 651–655 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Arcourt, A. et al. Touch receptor-derived sensory information alleviates acute pain signaling and fine-tunes nociceptive reflex coordination. Neuron 93, 179–193 (2017).

    CAS  PubMed  Google Scholar 

  43. Yamanaka, N. et al. Neuroendocrine control of Drosophila larval light preference. Science 341, 1113–1116 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Terada, S. et al. Neuronal processing of noxious thermal stimuli mediated by dendritic Ca(2+) influx in Drosophila somatosensory neurons. Elife 5, e12959 (2016).

    PubMed  PubMed Central  Google Scholar 

  45. Luo, J., Shen, W.L. & Montell, C. TRPA1 mediates sensation of the rate of temperature change in Drosophila larvae. Nat. Neurosci. 20, 34–41 (2017).

    CAS  PubMed  Google Scholar 

  46. Honjo, K., Mauthner, S.E., Wang, Y., Skene, J.H.P. & Tracey, W.D. Jr. Nociceptor-enriched genes required for normal thermal nociception. Cell Rep. 16, 295–303 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Lee, K.S., You, K.H., Choo, J.K., Han, Y.M. & Yu, K. Drosophila short neuropeptide F regulates food intake and body size. J. Biol. Chem. 279, 50781–50789 (2004).

    CAS  PubMed  Google Scholar 

  48. Shang, Y. et al. Short neuropeptide F is a sleep-promoting inhibitory modulator. Neuron 80, 171–183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Root, C.M., Ko, K.I., Jafari, A. & Wang, J.W. Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search. Cell 145, 133–144 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Solway, B., Bose, S.C., Corder, G., Donahue, R.R. & Taylor, B.K. Tonic inhibition of chronic pain by neuropeptide Y. Proc. Natl. Acad. Sci. USA 108, 7224–7229 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Han, C., Jan, L.Y. & Jan, Y.-N.N. Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila. Proc. Natl. Acad. Sci. USA 108, 9673–9678 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Song, W., Onishi, M., Jan, L.Y. & Jan, Y.N. Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae. Proc. Natl. Acad. Sci. USA 104, 5199–5204 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Parrish, J.Z., Kim, M.D., Jan, L.Y. & Jan, Y.N. Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites. Genes Dev. 20, 820–835 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Shearin, H.K., Dvarishkis, A.R., Kozeluh, C.D. & Stowers, R.S. Expansion of the gateway multisite recombination cloning toolkit. PLoS One 8, e77724 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Hughes, C.L. & Thomas, J.B. A sensory feedback circuit coordinates muscle activity in Drosophila. Mol. Cell. Neurosci. 35, 383–396 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Yang, C.H., Belawat, P., Hafen, E., Jan, L.Y. & Jan, Y.N. Drosophila egg-laying site selection as a system to study simple decision-making processes. Science 319, 1679–1683 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Han, C. et al. Integrins regulate repulsion-mediated dendritic patterning of drosophila sensory neurons by restricting dendrites in a 2D space. Neuron 73, 64–78 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Jiang, N., Soba, P., Parker, E., Kim, C.C. & Parrish, J.Z. The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth. Development 141, 2657–2668 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Leiss, F. et al. Characterization of dendritic spines in the Drosophila central nervous system. Dev. Neurobiol. 69, 221–234 (2009).

    CAS  PubMed  Google Scholar 

  61. Berger-Müller, S. et al. Assessing the role of cell-surface molecules in central synaptogenesis in the Drosophila visual system. PLoS One 8, e83732 (2013).

    PubMed  PubMed Central  Google Scholar 

  62. Christiansen, F. et al. Presynapses in Kenyon cell dendrites in the mushroom body calyx of Drosophila. J. Neurosci. 31, 9696–9707 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Potter, C.J., Tasic, B., Russler, E.V., Liang, L. & Luo, L. The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141, 536–548 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Gordon, M.D. & Scott, K. Motor control in a Drosophila taste circuit. Neuron 61, 373–384 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Baines, R.A., Uhler, J.P., Thompson, A., Sweeney, S.T. & Bate, M. Altered electrical properties in Drosophila neurons developing without synaptic transmission. J. Neurosci. 21, 1523–1531 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Grönke, S., Clarke, D.-F., Broughton, S., Andrews, T.D. & Partridge, L. Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 6, e1000857 (2010).

    PubMed  PubMed Central  Google Scholar 

  67. Hong, S.H. et al. Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals. PLoS Genet. 8, e1002857 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Lee, K.S. et al. Processed short neuropeptide F peptides regulate growth through the ERK-insulin pathway in Drosophila melanogaster. FEBS Lett. 583, 2573–2577 (2009).

    CAS  PubMed  Google Scholar 

  69. Potter, C.J. & Luo, L. Splinkerette PCR for mapping transposable elements in Drosophila. PLoS One 5, e10168 (2010).

    PubMed  PubMed Central  Google Scholar 

  70. Pfeiffer, B.D., Truman, J.W. & Rubin, G.M. Using translational enhancers to increase transgene expression in Drosophila. Proc. Natl. Acad. Sci. USA 109, 6626–6631 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Groth, A.C., Fish, M., Nusse, R. & Calos, M.P. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166, 1775–1782 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Luan, H., Peabody, N.C., Vinson, C.R. & White, B.H. Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron 52, 425–436 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Pfeiffer, B.D. et al. Refinement of tools for targeted gene expression in Drosophila. Genetics 186, 735–755 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Patel, N.H., Snow, P.M. & Goodman, C.S. Characterization and cloning of fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila. Cell 48, 975–988 (1987).

    CAS  PubMed  Google Scholar 

  75. Iwai, Y. et al. Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the Drosophila embryonic CNS. Neuron 19, 77–89 (1997).

    CAS  PubMed  Google Scholar 

  76. Wagh, D.A. et al. Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49, 833–844 (2006).

    CAS  PubMed  Google Scholar 

  77. Johard, H.A.D. et al. Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters. J. Comp. Neurol. 507, 1479–1496 (2008).

    CAS  PubMed  Google Scholar 

  78. Lin, D.M., Fetter, R.D., Kopczynski, C., Grenningloh, G. & Goodman, C.S. Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation. Neuron 13, 1055–1069 (1994).

    CAS  PubMed  Google Scholar 

  79. Honjo, K., Hwang, R.Y. & Tracey, W.D. Jr. Optogenetic manipulation of neural circuits and behavior in Drosophila larvae. Nat. Protoc. 7, 1470–1478 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Kernan, M., Cowan, D. & Zuker, C. Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila. Neuron 12, 1195–1206 (1994).

    CAS  PubMed  Google Scholar 

  81. Risse, B. et al. FIM, a novel FTIR-based imaging method for high throughput locomotion analysis. PLoS One 8, e53963 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Stocks obtained from the Bloomington (NIH P40OD018537), VDRC and Kyoto Drosophila Stock Centers were used in this study. Antibodies were obtained from the Developmental Studies Hybridoma Bank (created by the NICHD/NIH, maintained at The University of Iowa). We thank S. Sigrist for UAS-Brp-short-mCherry and UAS-Dα7-GFP, M. Gallio for the UAS-spGFP1-10-Syb, G. Tavosanis for the LexOP-brp-short-mCherry and K. Yu for sNPF and sNPF-R lines; J. Veenstra for sNPF antibodies; D. Pauls for discussion and reagents; T. Oertner, S. Wiegert, D. Pauls, J. Parrish, S. Zhu, C. Han, K. Duncan and F. Calderon for critical reading of the manuscript; and W. Grueber, A. Cardona and M. Zlatic for communicating results before publication. This work was supported by a US National Institutes of Health (NIH) grant R01GM100027 (to A.G. and C.H.Y.), the Landesförschungsförderung LFF-FV27 (to. P.S.) and the Deutsche Forschungsgemeinschaft priority program SPP1926, project SO1337/2-1 (to P.S.).

Author information

Authors and Affiliations

Authors

Contributions

The study was designed by C.H., M.P., N.H. and P.S. and was directed and coordinated by P.S. Morphological analyses were performed by C.H., M.P., B.S., F.T. and A.G. The behavioral experiments were performed by C.H., N.H., M.P., B.S., F.T. and L.S.B. Calcium imaging was performed by C.H. EM analysis was performed by D.W., F.T., E.S. and M.S. Transgenic lines were generated by M.P., A.R.G. and C.-H.Y. P.S. prepared the manuscript with input from all authors.

Corresponding author

Correspondence to Peter Soba.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of A08n neuron connectivity

(a) Co-labeling of C4da (ppk-CD4-tdTomato) and A08n neurons (6.14.3-Gal4, UAS-CD4-tdGFP). C4da axon terminals overlap with ladder-like projections of A08n neurons. A08n somata and additional neurons labeled by 6.14.3-Gal4 are indicated by arrows. Scale bar, 50 μm. (b) Flip-out labeling of 6.14.3-Gal4 expressing cells (UAS-CD4-tdTomato) shows that ladder-like projections are specific to A08n neurons. Scale bars, 50 μm. (c) A08n neurons were co-labeled by 6.14.3-Gal4 and 82E12-LexA expressing CD4-tdGFP and CD4-tdTomato, respectively. A08n somata are indicated by arrows. Scale bar: 50μm. (d) A08n split-Gal4 (82E12-Gal4AD, 6.14.3-Gal4DBD, UAS-CD4-tdGFP) expression pattern and co-labeling with C4da axon terminals (ppk-CD4-tdTomato). A08n somata and additionally weakly labeled A01b neurons are indicated by arrows. Scale bar, 50 μm. (e) Model of A08n and C4da neuron innervation A08n and synaptic overlap in VNC cross-sections. (f) A08n neurons expressing presynaptic Brp-short-mCherry and postsynaptic Dα7-GFP markers. Scale bar: 30 μm. (g) Syb-GRASP control. Expression of spGFP1-10-Syb in C4da neurons alone shows no reconstituted GFP signal. Scale bar, 25 μm.

Supplementary Figure 2 Characterization of DP-ilp7 neuron connectivity

(a) Co-labeling of C4da axon terminals (ppk-CD4-tdTomato) with ilp7 neurons (ilp7-Gal4, UAS-CD4-tdGFP) with arrows indicating DP-ilp7 somata. Scale bar, 50 μm. (b,b’) DP-ilp7 neuron morphology visualized by Flp-mediated CD4-tdTomato labeling together with Fas3-labelled sensory axons (C2da-C4da). Scale bar, 25 μm. (b’) Enlarged view of boxed area in (a) and resliced maximal projections along the YZ and XZ axis (indicated by dotted lines) show overlap of DP-ilp7 neurites with anti-Fas3 labeled sensory terminals. Scale bars, 5 μm. (c) Schematic projection patterns of sensory da neurons and DP-ilp7 neurons is shown. Boxed region is displays enlarged dorsal view (top panel) and cross-section view (bottom panel) indicating sites of sensory and DP-ilp7 neuron contact in ventromedial and -lateral regions of the VNC neuropil. (d) ilp7 neurons expressing presynaptic Brp-short-mCherry and postsynaptic Dα7-GFP markers together with anti-Fas3 labeling. Scale bar, 25 μm. (d’) Corresponding 3D-view of the boxed regions in (d) shows specific overlap of DP-ilp7 postsynaptic sites with both ventro-medial and -lateral Fas3 positive sensory terminals. Scale bar, 5 μm. (e) Syb-GRASP control. Expression of spGFP1-10-Syb in sensory da neurons (2-17-Gal4) alone shows no reconstituted GFP signal. Scale bar: 25 μm. (f) Pan-da neuron expression of CD4-tdGFP (21-7-Gal4) shows sensory terminal overlap with DP-ilp7 neurons (ilp7-LexA, LexOP-CD4-tdTomato). XY maximum projections and XZ projections of indicated regions (dotted lines) are shown. Scale bars, 10 μm.

Supplementary Figure 3 Analysis of nociceptive circuit elements in larval locomotion and innocuous-touch response

(a) Larval locomotion analysis of freely moving larvae expressing Kir2.1 in C4da, A08n or ilp7 neurons and controls. Animals were video-captured for 1 min and average locomotion speed was analyzed. A mild increase in locomotion speed was observed for C4da neuron silencing with Kir2.1 compared to Gal4 control, but not for any other tested neuronal subsets (n as indicated, p<0.001, p>0.05, Mann-Whitney U test). (b) Cumulative gentle touch scores for C2/3da, C4da, A08n and ilp7 driver lines expressing Kir2.1. C2/3da neuron inactivation decreased gentle touch behavior (n as indicated, p<0.0001, Mann-Whitney U test), while silencing of other subsets showed no defect in innocuous touch behavior (n as indicated, p>0.05, Mann-Whitney U test). (b’) Touch response scores according to Kernan et al.79.

Supplementary Figure 4 Functional analysis of A08n and ilp7 neuron connectivity and behavior

(a-b) Expression pattern of CsChrimson-GFP using (a) ppk-Gal4 or (b) 27H06-LexA in the (a,b) periphery and (a’,b’) CNS. High level expression of CsChrimson with ppk-Gal4 resulted in partial dendrite and axon degeneration of C4da neurons. Scale bars, 50 μm. (c-f) A08n neurons are sufficient to elicit nociceptive responses. Flipase-induced mosaic expression of CsChrimson-GFP in only one or few neurons. 3rd instar larvae were tested for light induced nociceptive behavior and subdivided into rollers/non-rollers. Larval brains of each group were dissected and examined for their expression pattern. (c) Schematic larval brain with expression pattern of 82E12-Gal4 showing the nomenclature used for labeled cell clusters. (d) Representative images of larval brains dissected after behavioral analysis. CsChrimson-GFP expressing neurons of larvae displaying no reaction (“non-rollers”) or nocifensive rolling (“rollers”) after activation were identified and assigned according to (c). Scale bars, 50 μm. (e) Percentage of animals displaying non- vs. rolling with particular cells expressing CsChrimson. Labeling of A08n neurons was significantly enriched in rolling vs. non-rolling larvae (p<0.001, two-tailed unpaired Student’s t-test). All other neurons except s2 and l2 showed no enrichment in labeling in rolling larvae (p>0.05, two-tailed unpaired Student’s t-test). (f) To exclude a role for s2 and l2 cells, the number of larvae showing light induced rolling (“rollers”) labeling A08n plus the indicated cell types showed that s2 and l2 cells are not correlated with nocifensive responses, but were co-expressing CsChrimson together with A08n. (g) Kir2.1 mediated inactivation of C4da neurons did not impair A08n induced optogenetic rolling responses (n as indicated, p>0.05, χ 2 test). (h) Rolling induced by optogenetic activation of A08n neurons was not affected by co-activation or silencing of ilp7 neurons (n as indicated, p>0.05, χ2 test). (i) Syb-GRASP between ilp7 presynaptic sites and A08n neurons shows no reconstituted GFP signal. Scale bar, 25 μm. (j) Syb-GRASP between A08n presynaptic sites and ilp7 neurons shows reconstituted GFP signal along the medial axon projections of A08n neurons. Scale bar, 25 μm. (j’) Enlarged view from (j’). Scale bar, 10 μm. (k) Syb-GRASP control. Expression of spGFP1-10-Syb in A08n neurons alone shows no reconstituted GFP signal. Scale bar, 25 μm. (l) Optogenetic activation of A08n or ilp7 neurons does not result in calcium responses of DP-ilp7 or A08n neurons respectively (n as indicated, p>0.05, Mann-Whitney U test).

Supplementary Figure 5 Behavioral analyses of DP-ilp7 and innocuous-touch neuron function

(a) CsChrimson-GFP expression pattern using DP-ilp7-LexA shows specific signal in DP-ilp7 neurons only. Scale bar: 50 μm. (b) Characterization of C2da neuron specific expression of R78A01-Gal4 using UAS-CD4-tdGFP together with ppk-CD4-tdTomato co-labeling C4da neurons. C2da neuron specific axon terminals are labeled in abdominal segments. Thoracic segments show partial labeling of C1da neuron projections. Arrows indicate medial terminal projections, arrowheads lateral, C2da neuron specific terminal projections. (b’) R78A01-Gal4 expresses in a subset of C2da neurons located on the dorsal (ddaB) and ventral (ldaA) body wall. However, the two ventral C2da neurons are not labeled. Scale bars, 30 μm. (c) Mechanonociceptive behavior of 3rd instar larvae at 96h AEL after the first (I) and second (II) mechanical stimulation with a 45 mN von Frey filament. Kir2.1 mediated silencing of C3da or C2/3da neurons inhibits mechano-nociceptive responses (n as indicated, p<0.001, χ2 test). (d) Tetanus toxin light chain (TnT) mediated silencing of C2/3da or A08n neurons strongly inhibits mechano-nociceptive responses (n as indicated, p<0.001, χ 2 test). (e) Tetanus toxin light chain (TnT) mediated silencing of C2/3da but not C2da neurons alone impairs innocuous touch behavior (n as indicated, p<0.001, Mann-Whitney U test). (f) Animals lacking ilp7 peptide (ilp7ko) show significantly increased mechano-nociceptive responses after the 1st stimulus (p<0.001, chi2 test) but not after the 2nd stimulus (n as indicated, p>0.05, χ 2 test).

Supplementary Figure 6 Ilp7 neuron-derived sNPF is required for mechanonociception

(a) sNPF is expressed in DP-ilp7 but not in other ilp7 neurons. The sNPF expression pattern was visualized with a MIMIC enhancer trap expressing GFP under control of the endogenous sNPF promoter (sNPFMI01807). Co-labeling of ilp7 neurons (ilp7-LexA,LexAop-CD4-tdTomato) showed overlapping expression in DP-ilp7 (arrowheads) but not posterior ilp7 neurons (arrows). Scale bar, 50 μm. (b) Anti-sNPF and anti-Fas3 staining of 3rd instar larval brain preparations of w1118 control, sNPFc00448 or sNPFMi01807 homozygous animals showed strongly reduced sNPF levels in both mutant alleles. Scale bar, 50 μm. (c) sNPF-RNAi in ilp7 but not C4da neurons impairs mechano-nociceptive responses (n as indicated, p<0.05, χ2 test). (d) Heterozygous sNPF mutant animals (sNPFc00448) show impaired mechano-nociceptive responses which are enhanced by ilp7 neuron specific sNPF knockdown by RNAi. Expression of sNPF1/2 in ilp7 neurons partially rescues rolling behavior compared to sNPF-RNAi (n as indicated, p<0.05, χ2 test). (e) Inhibition of sNPF-R function in C2/3da or C4da neurons expressing a dominant negative variant did not impair innocuous touch responses (n as indicated, p>0.05, Mann-Whitney U test). (f) Inhibition of sNPF-R function in basin neurons using RNAi mediated knockdown or a dominant negative variant did not impair mechano-nociceptive responses (n as indicated, p>0.05, χ2 test).

Supplementary Figure 7 Modality-specific requirement of DP-ilp7 neuron activity and sNPF function in mechanonociception

(a) Latency of nociceptive rolling responses was measured in 3rd instar larvae at 96h AEL after local simulation with a probe heated to 46 °C. Kir2.1 mediated silencing of ilp7 neurons does not induce thermo-nociceptive defects (n as indicated, p>0.05, Kruskal-Wallis ANOVA). (b) Inhibition of sNPF-R function in C4da neurons expressing a dominant negative variant did not significantly impair thermo-nociceptive responses (n as indicated, p>0.05, Kruskal-Wallis ANOVA). (c) Schematic of testing activity and sNPF function in C4da presynaptic and A08n neuron calcium responses. (c’) shows larval preparation used for calcium imaging with optical access to the VNC and von Frey filament stimulation of an abdominal segment (a4) on the intact posterior body wall. (d) Mechano-nociceptive calcium responses of A08n neurons (82E12-Gal4, UAS-GCaMP6m) after mechanical stimulation of abdominal segment 4-6 (45 mN) with and without ilp7 neuron inhibition by Kir2.1. (d’) Maximum responses (ΔFmax/F0) were plotted and compared (n=11/genotype, p<0.05, Mann-Whitney test). (e) Modality specific nociceptive circuit function requires distinct circuit and neuromodulatory components. Harsh mechanical touch activates C2-C4da neurons, which results in activation of DP-ilp7 neurons (indicated by color coded arrows). sNPF derived from DP-ilp7 neurons is required for paracrine feedback signaling via sNPF-R in C2/3/4da neurons (light blue arrows). sNPF-R signaling in turn results in C4da neuron presynaptic facilitation and successive increase in A08n response to trigger nociceptive escape behavior. Thermo-nociceptive behavior is likely encoded by additional circuit components independent of the identified network elements. (f) Synaptic model of sNPF and sNPF-R action. Activity results in local release of sNPF from DP-ilp7 neurons. Released sNPF can then activate sNPF-Rs expressed in sensory C2/C3/C4da neurons in a paracrine fashion.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1. (PDF 2042 kb)

Supplementary Methods Checklist (PDF 231 kb)

A08n response to C4da neuron activation

A08n Calcium response after light induced C4da neuron activation (6.14.3-Gal4, UAS-GCaMP6m;27H06-LexA, LexAop-CsChrimson). Note that local interneurons labeled by 6.14.3-Gal4 are not activated by the 625nm light stimulus. (AVI 3278 kb)

Optogenetic activation of C4da neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in C4da neurons (27H06-LexA, LexAop-CsChrimson) were exposed to 625nm light for 5s which induced larval rolling, writhing and locomotion speedup. (AVI 14234 kb)

Optogenetic activation of A08n neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in A08n neurons (6.14.3-Gal4, UAS-CsChrimson) were exposed to 625nm light for 5s which recapitulates the C4da induced nocifensive response. (AVI 14041 kb)

DP-ilp7response to C1da-C4da neuron activation

DP-ilp7 Calcium response after light induced C1-C4da neuron activation (21-7-Gal4>CsChrimson;ilp7-LexA>GCaMP6m). (AVI 374 kb)

Optogenetic activation of C2da neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in C2da neurons (ppk-Gal80; R78A01-Gal4,UAS-CsChrimson) were exposed to 625nm light for 5s which induced C-shaped bending and slow larval rolling without writhing and locomotion speedup. (AVI 8280 kb)

Optogenetic activation of C3da neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in C3da neurons (tsh-Gal80;19-12-Gal4,UAS-CsChrimson) were exposed to 625nm light for 5s which induced contraction (“hunching”) of all body segments. (AVI 4740 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hu, C., Petersen, M., Hoyer, N. et al. Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci 20, 1085–1095 (2017). https://doi.org/10.1038/nn.4580

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.4580

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing