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.

  • Article
  • Published:

TRPA1 mediates sensation of the rate of temperature change in Drosophila larvae

Nature Neuroscience volume 20, pages 34–41 (2017)Cite this article

Subjects

Abstract

Avoidance of noxious ambient heat is crucial for survival. A well-known phenomenon is that animals are sensitive to the rate of temperature change. However, the cellular and molecular underpinnings through which animals sense and respond much more vigorously to fast temperature changes are unknown. Using Drosophila larvae, we found that nociceptive rolling behavior was triggered at lower temperatures and at higher frequencies when the temperature increased rapidly. We identified neurons in the brain that were sensitive to the speed of the temperature increase rather than just to the absolute temperature. These cellular and behavioral responses depended on the TRPA1 channel, whose activity responded to the rate of temperature increase. We propose that larvae use low-threshold sensors in the brain to monitor rapid temperature increases as a protective alert signal to trigger rolling behaviors, allowing fast escape before the temperature of the brain rises to dangerous levels.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Rolling responses of wild-type larvae exposed to different rates of temperature increase.
Figure 2: Effects of eliminating different thermoTRPs on Frolling values in response to the same dT/dt.
Figure 3: Effects of eliminating different TRPA1 isoforms on Frolling values in response to dT/dt.
Figure 4: Identifying trpA1-AB neurons in the larval brain required for heat-induced rolling.
Figure 5: Relative expression of trpA1-AB and trpA1-CD in body wall and CNS of third-instar larvae.
Figure 6: Effects of dT/dt on the activities of BLP and BLA neurons of third-instar larvae.
Figure 7: Effects of dT/dt on the activities of the TRPA1-A and TRPA1-D channels.

Similar content being viewed by others

References

  1. Julius, D. TRP channels and pain. Annu. Rev. Cell Dev. Biol. 29, 355–384 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Patapoutian, A., Peier, A.M., Story, G.M. & Viswanath, V. ThermoTRP channels and beyond: mechanisms of temperature sensation. Nat. Rev. Neurosci. 4, 529–539 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Venkatachalam, K. & Montell, C. TRP channels. Annu. Rev. Biochem. 76, 387–417 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Venkatachalam, K., Luo, J. & Montell, C. Evolutionarily conserved, multitasking TRP channels: lessons from worms and flies. Handb. Exp. Pharmacol. 223, 937–962 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Caterina, M.J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Caterina, M.J. et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306–313 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Bautista, D.M. et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204–208 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. McKemy, D.D., Neuhausser, W.M. & Julius, D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416, 52–58 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Peier, A.M. et al. A TRP channel that senses cold stimuli and menthol. Cell 108, 705–715 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Story, G.M. et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112, 819–829 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Xiao, R. et al. A genetic program promotes C. elegans longevity at cold temperatures via a thermosensitive TRP channel. Cell 152, 806–817 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  13. Lee, Y. et al. Pyrexia is a new thermal transient receptor potential channel endowing tolerance to high temperatures in Drosophila melanogaster. Nat. Genet. 37, 305–310 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Neely, G.G. et al. TrpA1 regulates thermal nociception in Drosophila. PLoS One 6, e24343 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kwon, Y., Shim, H.S., Wang, X. & Montell, C. Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade. Nat. Neurosci. 11, 871–873 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Shen, W.L. et al. Function of rhodopsin in temperature discrimination in Drosophila. Science 331, 1333–1336 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Sedgwick, W.T. On variations of reflex-excitability in the frog, induced by changes of temperature. Stud. Biol. Lab., Johns Hopkins University 385 (1882).

  19. Clark, D.A., Gabel, C.V., Lee, T.M. & Samuel, A.D. Short-term adaptation and temporal processing in the cryophilic response of Caenorhabditis elegans. J. Neurophysiol. 97, 1903–1910 (2007).

    Article  PubMed  Google Scholar 

  20. Green, B.G. & Akirav, C. Threshold and rate sensitivity of low-threshold thermal nociception. Eur. J. Neurosci. 31, 1637–1645 (2010).

    PubMed  PubMed Central  Google Scholar 

  21. Gershow, M. et al. Controlling airborne cues to study small animal navigation. Nat. Methods 9, 290–296 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mitchell, T.M. Machine Learning (McGraw-Hill, 1997).

  23. Manning, C.D., Raghavan, P. & Schütze, H. Introduction to Information Retrieval (Cambridge University Press, Cambridge, 2008).

  24. Viswanath, V. et al. Opposite thermosensor in fruitfly and mouse. Nature 423, 822–823 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Sokabe, T., Tsujiuchi, S., Kadowaki, T. & Tominaga, M. Drosophila painless is a Ca2+-requiring channel activated by noxious heat. J. Neurosci. 28, 9929–9938 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Babcock, D.T. et al. Hedgehog signaling regulates nociceptive sensitization. Curr. Biol. 21, 1525–1533 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Oswald, M., Rymarczyk, B., Chatters, A. & Sweeney, S.T. A novel thermosensitive escape behavior in Drosophila larvae. Fly (Austin) 5, 304–306 (2011).

    Article  Google Scholar 

  28. Ni, L. et al. A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila. Nature 500, 580–584 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kang, K. et al. Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila. Nature 481, 76–80 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kim, S.H. et al. Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons. Proc. Natl. Acad. Sci. USA 107, 8440–8445 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Kondo, S. & Ueda, R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics 195, 715–721 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yu, Z. et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195, 289–291 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gratz, S.J. et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 194, 1029–1035 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bassett, A.R., Tibbit, C., Ponting, C.P. & Liu, J.L. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 4, 220–228 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang, Y.Y., Chang, R.B., Waters, H.N., McKemy, D.D. & Liman, E.R. The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions. J. Biol. Chem. 283, 32691–32703 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Jordt, S.E. et al. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427, 260–265 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Nagata, K., Duggan, A., Kumar, G. & García-Añoveros, J. Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J. Neurosci. 25, 4052–4061 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zurborg, S., Yurgionas, B., Jira, J.A., Caspani, O. & Heppenstall, P.A. Direct activation of the ion channel TRPA1 by Ca2+. Nat. Neurosci. 10, 277–279 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Stoney, S.D. Jr. & Machne, X. Mechanisms of accommodation in different types of frog neurons. J. Gen. Physiol. 53, 248–262 (1969).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Hamada, F.N. et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature 454, 217–220 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kwon, Y. et al. Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal. Curr. Biol. 20, 1672–1678 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Bellen, H.J. et al. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167, 761–781 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Berni, J., Pulver, S.R., Griffith, L.C. & Bate, M. Autonomous circuitry for substrate exploration in freely moving Drosophila larvae. Curr. Biol. 22, 1861–1870 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Shearin, H.K., Macdonald, I.S., Spector, L.P. & Stowers, R.S. Hexameric GFP and mCherry reporters for the Drosophila GAL4, Q, and LexA transcription systems. Genetics 196, 951–960 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Duffy, J.B., Harrison, D.A. & Perrimon, N. Identifying loci required for follicular patterning using directed mosaics. Development 125, 2263–2271 (1998).

    CAS  PubMed  Google Scholar 

  51. Inagaki, H.K. et al. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat. Methods 11, 325–332 (2014).

    Article  CAS  PubMed  Google Scholar 

  52. Taghert, P.H. et al. Multiple amidated neuropeptides are required for normal circadian locomotor rhythms in Drosophila. J. Neurosci. 21, 6673–6686 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Pfeiffer, B.D. et al. Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. USA 105, 9715–9720 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Ni, J.Q. et al. A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182, 1089–1100 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ohyama, T. et al. High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies. PLoS One 8, e71706 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ben-Ialli, A., Bohuon, P., Collignan, A. & Méot, J.-M. Modeling heat transfer for disinfestation and control of insects (larvae and eggs) in date fruits. J. Food Eng. 116, 505–514 (2013).

    Article  Google Scholar 

  57. Gong, W.J. & Golic, K.G. Ends-out, or replacement, gene targeting in Drosophila. Proc. Natl. Acad. Sci. USA 100, 2556–2561 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Moon, S.J., Lee, Y., Jiao, Y. & Montell, C. A Drosophila gustatory receptor essential for aversive taste and inhibiting male-to-male courtship. Curr. Biol. 19, 1623–1627 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. DeCoursey, T.E. & Cherny, V.V. Temperature dependence of voltage-gated H+ currents in human neutrophils, rat alveolar epithelial cells, and mammalian phagocytes. J. Gen. Physiol. 112, 503–522 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Klein, M. et al. Sensory determinants of behavioral dynamics in Drosophila thermotaxis. Proc. Natl. Acad. Sci. USA 112, E220–E229 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  62. Gracheva, E.O. et al. Molecular basis of infrared detection by snakes. Nature 464, 1006–1011 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Macdonald (UC Santa Barbara) and H. Luo (Shanghai Jiao Tong University) for assistance in generating the knock-in fly lines; J. Liu and H. Chen (UC Santa Barbara) for assistance in performing blind optogenetic experiments; B. Afonso (Janelia Research Campus), M. Zlatic (Janelia Research Campus), M. Gershow (Harvard University) and A.D.T. Samuel (Harvard University) for help building the software and hardware for the larval tracking system; W.D. Tracey (Indiana University) for trpA1-BAC (ref. 14); K. Scott (UC Berkeley) for GRASP flies36; P.A. Garrity (Brandeis University) for the pOX-trpA1-A construct43; and G.M. Rubin and J.W. Truman (Janelia Research Campus) for the expression data corresponding to the adult and larval Janelia GAL4 lines. W.L.S. was supported by National Nature Science Foundation of China (X-0402-14-002). This work was supported by grants to C.M. from the National Eye Institute (EY010852) and the National Institute on Deafness and Other Communication Disorders (DC007864).

Author information

Author notes

  1. Wei L Shen

    Present address: Present address: ShanghaiTech University, Shanghai, China.,

Authors and Affiliations

  1. Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California, USA

    Junjie Luo & Craig Montell

  2. Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, California, USA

    Junjie Luo & Craig Montell

  3. Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Junjie Luo & Wei L Shen

Authors
  1. Junjie Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wei L Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Craig Montell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The study was designed by J.L., W.L.S. and C.M., and directed and coordinated by C.M. The behavioral experiments were performed by J.L. and W.L.S. J.L. generated the trpA1 alleles and performed the immunohistochemistry experiments and Ca2+ imaging experiments. W.L.S. and J.L. performed two-electrode recordings in Xenopus oocytes. The manuscript was prepared by J.L., W.L.S. and C.M.

Corresponding author

Correspondence to Craig Montell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Rolling responses of trpA11 larvae exposed to different rates of temperature increase.

(a-i) The fraction of trpA11 second-instar larvae that rolled (Frolling) as a function of the rate of temperature change (dT/dt). The dT/dt are indicated above each plot. The scale bars indicate the seconds required for the temperature to rise by 5 °C. The rolling fraction is defined as Nrolling/Ntotal, where Nrolling is the number of larvae rolling and Ntotal is the total number of larvae. Fpeak is the maximum rolling fraction and Tmiddle is the temperature corresponding to the point when the rolling fraction rose to half the Fpeak. The curves (solid black lines) are fit using a sigmoid function. Total number of larvae per experiment ≥ 30. n = 4, 6, 5, 6, 4, 4, 7, 9 and 8 in a, b, c, d, e, f, g, h and i, respectively. (j) Fpeak values as a function of dT/dt. The crosses indicate the average Fpeak values in response to the different temperature changing rates. Error bars indicate s.e.m.

Supplementary Figure 2 Expression of trpA1-A using the GAL4/UAS system lowered the thermal nociception threshold.

Second-instar larvae were exposed to a heat ramp (dT/dt) of 0.1 °C/s. (a) Control (w1118) larvae. n = 3 (≥ 30 larvae per experiment). (b) Larvae expressing trpA1-A (UAS-trpA1-A) using the trpA1-ABGAL4 in a transheterozygous trpA1 mutant background (trpA11/trpA1-ABGAL4). n = 4 (≥ 30 larvae per experiment). (c) Expression of UAS-trpA1-B in a trpA1-ABGAL4/trpA11 background. n = 3 (≥ 30 larvae per experiment). (d) Expression of trpA1-A (UAS-trpA1-A) in a trpA1-ABGAL4/+ heterozygous larvae. n = 3 (≥ 30 larvae per experiment).

Supplementary Figure 3 Rolling responses of larvae ectopically expressing trpA1-A in mdIV neurons exposed to different rates of temperature increase.

(a-i) The fraction of second-instar larvae that rolled (Frolling) as a function of the rate of temperature change (dT/dt). Blue, ppk-GAL4/UAS-trpA1-A;trpA11 (Total number of larvae per experiment ≥ 30. n = 3, 3, 6, 3, 3, 3, 4, 3 and 5 in a, b, c, d, e, f, g, h and i, respectively). Red, UAS-trpA1-A;trpA11 (Total number of larvae per experiment ≥ 30. n = 4, 3, 5, 3, 3, 3, 4, 3 and 5 in a, b, c, d, e, f, g, h and i, respectively). Green, ppk-GAL4;trpA11, only in a, e and i (Total number of larvae per experiment ≥ 30. n = 3, 4 and 4 in a, e and i respectively). The dT/dt are indicated above each plot. The scale bars indicate the seconds required for the temperature to rise by 5 °C. The rolling fraction was defined as Nrolling/Ntotal, where Nrolling was the number of larvae rolling and Ntotal was the total number of larvae. Fpeak was the maximum rolling fraction and Tmiddle was the temperature corresponding to the point when the rolling fraction rose to half the Fpeak. The curves (solid black lines) were fit using a sigmoid function. (j) Fpeak values as a function of dT/dt. Crosses, average Fpeak values in response to the different temperature-change rates. (k) Tmiddle values as a function of dT/dt. Crosses, average Tmiddle values. ppk-GAL4/UAS-trpA1-A;trpA11, one-way ANOVA of Fpeak: F8,24 = 17.81; P = 2.4 x 10-8; one-way ANOVA of Tmiddle: F8,24 = 1.79; P = 0.13; Tukey-Kramer test for statistically significant differences relative to the highest dT/dt (0.5 °C/s): Fpeak: 0.5 °C/s versus. 0.02 °C/s: q24,9 = 8.77; P = 6.3 x 10-5. *P < 0.05, **P < 0.01. Error bars indicate s.e.m.

Supplementary Figure 4 Expression of the trpA1-AB and trpA1-CD reporters in the CNS of third-instar larvae.

(a) An enlarged version of Fig. 5b. (n = 16). The larval genotype was UAS-GFP/+;trpA1-ABLexA,LexAop-frt-mCherry-STOP-frt-ReaChR::Citrine/trpA1-CDGAL4. The colored arrows indicate different trpA1-AB neuronal clusters. The first letter indicates whether the neurons were in the brain (B) or the VNC (V). The second letter indicates the general region within the brain or VNC that contained the neuronal cell bodies: A, anterior; C, central; L, lateral; P, posterior. The third letter indicates the relative positions of the neuronal clusters within the general regions of the brain and VNC: A, anterior; C, central; L, lateral; M, medial, P, posterior. The intensities of the BCA, and BCM neurons were not great enough to detect in this image. (b) An enlarged version of Fig. 4a in which the BCA and BCM neurons were bright enough to detect. (n = 7). The larval genotype was trpA1-ABLexA,LexAop-frt-mCherry-STOP-frt-ReaChR::Citrine/+. Scale bars, 50 μm.

Supplementary Figure 5 Expression of the trpA1-AB reporter in the CNS of a trpA1-ABLexA homozygous mutant third-instar larva.

The colored arrows indicate different trpA1-AB neuronal clusters. (n = 9). The larval genotype was trpA1-ABLexA,LexAop-frt-mCherry-STOP-frt-ReaChR::Citrine. The first letter indicates whether the neurons were in the brain (B) or the VNC (V). The second letter indicates the general region within the brain or VNC that contained the neuronal cell bodies: A, anterior; C, central; L, lateral; P, posterior. The third letter indicates the relative positions of the neuronal clusters within the general regions of the brain and VNC: A, anterior; C, central; L, lateral; M, medial, P, posterior. Scale bar (lower left), 50 μm.

Supplementary Figure 6 Overlap of the indicated GAL4 reporters with the trpA1-ABLexA/+ reporter in third-instar larvae.

The dashed lines outline the larval brain and VNC. The whole mounts were stained with anti-DsRed and anti-GFP. (a-f) In addition to the GAL4 and trpA1-ABLexA/+ reporters, the larvae carried UAS-FLP and a LexAop-frt-mCherry-STOP-frt-ReaChR::Citrine transgenes. (g-i) In addition to the GAL4 and trpA1-ABLexA/+ reporters, the larvae carried UAS-GFP and a LexAop-frt-mCherry-STOP-frt-ReaChR::Citrine transgenes. Scale bars, 50 μm. (R60F07-GAL4, n = 5; 386Y-GAL4, n = 6; tsh-GAL80;trpA1-ABGAL4, n = 5).

Supplementary Figure 7 Overlap of the indicated GAL4 reporters with the trpA1-ABLexA/+ reporter in third-instar larvae.

In addition to the GAL4 and trpA1-ABLexA/+ reporters, the flies carried UAS-GFP and LexAop-frt-mCherry-STOP-frt-ReaChR::Citrine transgenes. The dashed lines outline the larval brain and VNC. The whole mounts were stained with anti-DsRed and anti-GFP. Scale bars, 50 μm. (R21E09-GAL4, n = 4; R21G01-GAL4, n = 5; R21F01-GAL4, n = 3).

Supplementary Figure 8 Effect on rolling behavior (Fpeak) resulting from knockdown of trpA1, using UAS-Dicer2;UAS-trpA1 RNAi and the indicated GAL4 drivers.

The animals were second-instar larvae. The temperature increased from 23.5 °C to 40 °C with a dT/dt = 0.1 °C/s. Total number of larvae per experiment ≥ 30. n = 9, 8, 4, 8, 3, 4 and 3 in a, b, c, d, e, f and g, respectively. Error bars indicate s.e.m.

Supplementary Figure 9 Temperature responses of BLP and BLA neurons of third-instar larvae in the presence of tetrodotoxin.

In order to address whether the temperature induced responses of BLP and BLA neurons (n = 30 and 9, respectively) were caused by presynaptic neurons, we applied 1 μM tetrodotoxin (TTX), which suppresses voltage-gated Na+ channels and synaptic transmission. These neurons still responded to temperature changes in the presence of TTX.

Supplementary Figure 10 Total current under slow and fast ramp conditions in oocytes expressing the TRPA1-A channel.

The total current was calculated by integrating the current during the slow and fast heat ramps (the areas under the current curves). We compared the total current under the slow and fast temperature ramps using the Wilcoxon signed rank test: n = 15, W = 120, P = 6.1 x 10-5).

Supplementary Figure 11 Apparatus for assaying larval thermal nociception behavior.

The black, plastic outer box (2’ x 2’ x 4’) eliminates external light. We used red (560 nm) LED lights to evenly illuminate the larvae placed on the agarose plate. The camera we used to monitor the larval behaviors was a 2592 x 1944 Monochrome CMOS Camera (GigE). We used a TC-36-25-RS232 temperature controller (TE Technology) to control the temperature changes of the 12” x 12” Peltier plates. We integrated the controller program with the larval tracking software.

Supplementary Figure 12 Comparison of the rolling behaviors of control (w1118) larvae at the indicated developmental stages (dT/dt = 0.2 °C/s).

(a) Second-instar larvae (n = 7; ≥ 30 larvae per experiment). (b) Third-instar larvae (n = 3; ≥ 30 larvae per experiment).

Supplementary Figure 13 Generation of the trpA1 alleles.

(a) The trpA1-ABLexA allele was generated using the CRISPR/Cas9 system, and trpA1-CDGAL4 was generated by ends-out homologous recombination. The cyan bars indicate the homologous arms in the constructs used to generate the knockouts. The purple, green and red polygons represent the LexA, GAL4 and mini-w genes, respectively, which were knocked into the genome. The gray and orange rectangles indicate the UTRs and trpA1 coding sequences, respectively. The purple and green triangles represent the primer pairs used to identify the trpA1-ABLexA and trpA1-CDGAL4 alleles, respectively. (b) Identification of trpA1-CDGAL4 by PCR analysis using the indicated primer pairs. (c) The identification of trpA1-ABLexA by PCR analysis using the indicated primer pairs. (d) The sequences encompassing the mutation in the trpA1-ACDGAL4 allele. The dashes indicate the two base pairs deleted in trpA1-ACDGAL4.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Luo, J., Shen, W. & Montell, C. TRPA1 mediates sensation of the rate of temperature change in Drosophila larvae. Nat Neurosci 20, 34–41 (2017). https://doi.org/10.1038/nn.4416

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.4416

This article is cited by

Search

Advanced 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