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