Circadian clock neurons constantly monitor environmental temperature to set sleep timing

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Abstract

Circadian clocks coordinate behaviour, physiology and metabolism with Earth’s diurnal cycle1,2. These clocks entrain to both light and temperature cycles3, and daily environmental temperature oscillations probably contribute to human sleep patterns4. However, the neural mechanisms through which circadian clocks monitor environmental temperature and modulate behaviour remain poorly understood. Here we elucidate how the circadian clock neuron network of Drosophila melanogaster processes changes in environmental temperature. In vivo calcium-imaging techniques demonstrate that the posterior dorsal neurons 1 (DN1ps), which are a discrete subset of sleep-promoting clock neurons5, constantly monitor modest changes in environmental temperature. We find that these neurons are acutely inhibited by heating and excited by cooling; this is an unexpected result when considering the strong correlation between temperature and light, and the fact that light excites clock neurons6. We demonstrate that the DN1ps rely on peripheral thermoreceptors located in the chordotonal organs7,8 and the aristae9. We also show that the DN1ps and their thermosensory inputs are required for the normal timing of sleep in the presence of naturalistic temperature cycles. These results identify the DN1ps as a major gateway for temperature sensation into the circadian neural network, which continuously integrates temperature changes to coordinate the timing of sleep and activity.

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Figure 1: Drosophila circadian clock neurons respond to temperature changes.
Figure 2: DN1ps are acutely excited by cooling and inhibited by heating.
Figure 3: Thermoreceptors in the aristae and the chordotonal organs are required for the responses of DN1ps to temperature changes.
Figure 4: DN1ps are required for the synchronization of behaviour to ramping temperature cycles.

Change history

  • 28 February 2018

    Source Data for Extended Data Fig. 7 was added.

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Acknowledgements

This work was supported by a Damon Runyon Cancer Foundation postdoctoral fellowship (DR2231-15) to S.Y., a National Science Foundation (NSF) CBET grant (1509691) to P.R. and E.M., a University of Michigan M-cubed grant to E.M., P.R. and O.T.S., and a National Institutes of Health NINDS grant (R01NS077933) and an NSF IOS grant (1354046) to O.T.S. We thank N. Glossop, P. Hardin, M. Rosbash and R. Stanewsky for fly stocks. We also thank M. Rosbash, L. Griffith and P. Garrity for discussions about our work and M. de la Paz Fernández for reading the manuscript.

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Authors

Contributions

The work was conceived by S.Y. and O.T.S. The calcium-imaging and behavioural experiments were performed by S.Y. and C.J., under the supervision of O.T.S. C.J. and S.Y. designed and built the Peltier and CaMPARI setups under the guidance of P.R. and E.M. S.Y., C.J. and A.B. conducted CaMPARI experiments. Data analysis was performed by S.Y. and C.J. The manuscript was written by S.Y., C.J. and O.T.S. with input from all authors.

Corresponding author

Correspondence to Orie T. Shafer.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks P. Emery, R. Stanewsky and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 CaMPARI protocol for the identification of temperature-sensitive clock neurons.

a, Schematic (left) and photograph (right) of the illumination and thermal control system for the CaMPARI experiments (see Methods). b, Temperature and LED illumination protocol for whole-fly CaMPARI experiments (see Methods). ce, Measured temperature of the Peltier element (c), and the fly during CaMPARI (d) and GCAMP (e) experiments. Heating and cooling stimuli were provided by changing the Peltier element set-point from 23 °C to 32 °C (red line) and from 23 °C to 15 °C (blue line), respectively (see Methods). fh, Representative confocal microscopy images of green and red CaMPARI fluorescence in the DN2s (f), l-LNvs (g), and LNds (h) in response to constant temperature (23 °C, top panel), heating (23 °C to 29 °C, middle panel), and cooling (23 °C to 16 °C, bottom panel). These experiments were repeated independently using seven brains with similar results.

Extended Data Figure 2 CaMPARI photoconversion in isolated brains.

a, Temperature stimuli and LED illumination protocol for CaMPARI experiments on isolated brains. In these experiments, the temperature was increased from 23 °C to 32 °C and only two cycles of illumination were used to avoid excessive photoconversion (see Methods). b, Quantification of the ratio of red and green CaMPARI fluorescence in clock neurons from isolated brains ectopically expressing the thermosensitive channel TRPA120, which is activated by temperatures greater than 25 °C. Clock neurons ectopically expressing TRPA1 display high levels of CaMPARI photoconversion in response to heating (grey bars, number of neurons: 5 DN1ps, 4 DN2s, 5 LNds) compared to constant temperature controls (white bars, number of neurons: 5 DN1ps, 5 DN2s, 6 LNds). c, Ratio of red to green CaMPARI fluorescence in clock neurons in isolated brains in response to cooling (white bars, 63 DN1ps, 9 DN2s, 7 DN3s, 19 LNds, 20 l-LNvs) and constant temperature (grey bars, 33 DN1ps, 10 DN2s, 10 DN3s, 26 LNds, 23 l-LNvs). Histograms represent the mean ratio of red to green fluorescence ± s.e.m., three to four brains analysed for each condition. Individual data points are shown, as the sample size is less than 10. **P < 0.005, unpaired two-tailed Student’s t-test. Specific P values are reported in the Source Data for this figure. Source data

Extended Data Figure 3 GCaMP protocol for the quantification of clock neuron responses to temperature changes.

a, b, Schematic of the GCaMP volume imaging (a) and quantification (b) during temperature changes (see Methods). c, Averaged LNd GCaMP6m fluorescence changes during cooling (black, n = 8 neurons) and at constant temperature (blue, n = 7). Averaged eGFP fluorescence traces from LNds during cooling (green, n = 9). These data were summarized as average maximum ΔF/F0 (%) increase ± s.e.m. in the histograms (right). LNds displayed no apparent GCaMP responses to cooling. d, Averaged LNd GCaMP6m fluorescence traces during heating (black, n = 12). Constant temperature GCaMP6m traces (blue, same data as in c) and eGFP traces during heating (green, n = 10 neurons) are also shown. These data were summarized as average minimum ΔF/F0 (%) ± s.e.m. in the histograms (right). The LNds appeared to display a loss of GCaMP fluorescence in response to heating; however, eGFP fluorescence displayed a similar response to heating (unpaired two-tailed Student’s t-test), revealing this response to be an artefact. e, Average DN1p GCaMP6m responses are proportional to the magnitude of the cold stimulus (top, n = 8) or hot stimulus (bottom, n = 26). f, Averaged DN1p GCaMP6m responses from wild-type (WT) flies at two different times during the diurnal cycle (black, ZT4–6, n = 10; green, ZT16–18, n = 5), and per01 flies at the same time points (red, ZT4–6, n = 7; blue, ZT16–18, n = 8). For the averaged GCaMP6m fluorescence traces, dark lines indicate the mean and shaded areas indicate the s.e.m. n = the number of LNds as stated in c, d and n = the number of DN1ps as stated in e, f. Individual data points are shown, as n is less than 10. Five to seven brains were analysed in each experiment. Temperature plots are displayed below the GCaMP traces. Specific P values are reported in the Source Data for this figure. Source data

Extended Data Figure 4 DN1p GCaMP responses in isolated brains, and in flies in which nocte is downregulated in the chordotonal organs.

a, b, Averaged DN1p GCaMP6m fluorescence traces during cooling (a) and heating (b), after flies were decapitated and aristae were removed, leaving the rest of the antennae and head cuticle intact. Data in the histograms are presented as average maximum increase (a) and decrease (b) of ΔF/F0 (%) ± s.e.m. for cooling and heating, respectively. Responses were not significantly different between heads from which aristae were removed (black, n = 7 in a, n = 11 in b) and isolated brains (blue, n = 32 in a, n = 25 in b). These data suggest that aristae are the only peripheral thermoreceptors on the head that contribute to DN1p temperature responses. The thermoreceptors of the sacculi, which remained after arista removal, are not sufficient for DN1p temperature responses, although it is possible that the cutting of the aristae may have had non-specific effects on saccular function. Unpaired two-tailed Student’s t-test. c, GCaMP6m responses of DN1ps in response to heating in isolated brains in HL3 solution (black, n = 25) and in HL3 supplemented with 2 μM tetrodotoxin and 400 μM CdCl2 to prevent neuronal firing and synaptic transmission (red, n = 20), and in the explanted brains of TRPA11 flies in HL3 (blue, n = 11). Histogram plotted as in b, ANOVA was performed across the three conditions (F2,55 = 1.395, P > 0.05) and Tukey’s honest significant difference tests were conducted in pairwise fashion with P values indicated on the histogram (right). These results suggest that the loss of GCaMP fluorescence seen in DN1ps in isolated brains in the absence of aristae and chordotonal organs is a non-physiological artefact. d, Averaged GCaMP3 fluorescence traces from DN1ps during cooling in control UAS-nocteRNAi1/+ flies (black, n = 6) and F-gal4>UAS-nocteRNAi1 flies (blue, n = 6) (F-Gal4 drives expression predominantly in chordotonal organs7). Histogram plotted as in a. e, Averaged GCaMP3 fluorescence traces from the DN1ps during heating in control UAS-nocteRNAi1/+ (black, n = 5) and F-gal4>UAS-nocte RNAi1 flies (red, n = 5). Histogram plotted as in b. For averaged GCaMP fluorescence traces, dark lines indicate mean and shaded areas indicate s.e.m. n = number of DN1ps, individual data points shown when n is less than 10. Four to six brains were analysed for each condition. **P < 0.005, unpaired two-tailed Student’s t-test. Individual P values are reported in the Source Data for this figure. Source data

Extended Data Figure 5 Activity and sleep profiles of wild-type Canton S flies under different amplitudes of ramping temperature cycles.

ag, Canton S flies were entrained to ramping temperature cycles of differing amplitudes and ranges: 18 °C to 28 °C or 22 °C to 28 °C for a week, followed by release into constant temperature conditions (25 °C) for another week. a, b, Averaged population activity of flies over the last four days of the temperature cycles (left) and averaged sleep plots under the same conditions (right). Dark lines indicate mean and shaded regions indicate s.e.m. n values are shown in d. c, Quantification of the anticipation of cooling for flies entrained to ramping temperature cycles from 18 °C to 25 °C, 18 °C to 28 °C and 22 °C to 28 °C. Data represent mean ± s.e.m. n values are shown in d. d, Summary of free-running locomotor activity rhythms of Canton S flies under constant conditions after entrainment to ramping temperature cycles. Data represent population average ± s.e.m. eg, Averaged population locomotor activity on the day in which the temperature was unpredictably decreased (e, f) or increased (g) (left) and averaged sleep profiles (right) (see Methods) (n = 32 (e), n = 61 (f), n = 63 (g)). Blue bars indicate unanticipated cooling, red bars indicate unanticipated heating. Dark lines indicate mean and shaded regions indicate s.e.m. Black dashed lines in the plots represent temperature. n = number of flies.

Extended Data Figure 6 Locomotor behaviour of wild-type and genetically manipulated flies under ramping temperature cycles and under constant conditions after entrainment.

a, Summary of heating index under ramping temperature cycles and free-running locomotor activity rhythms under constant conditions after entrainment to 18 °C to 25 °C ramping temperature cycles. per01, aristae-less nocte+ flies (nocte+ AL), aristae-less nocte1 mutants (nocte1AL) and Clk4.1m>UAS-TNT flies, in which synaptic transmission is blocked in DN1ps, displayed strong behavioural phenotypes during the heating phase of the temperature cycle as revealed by low correlation coefficients between activity and temperature during the heating phase (see Methods). per01, nocte1 mutants and flies lacking aristae displayed arrhythmic locomotor activity under constant conditions after entrainment to ramping temperature cycles. Data were presented as population average ± s.e.m. b, A summary of cooling indices (see Methods) for experimental and control flies. n values are shown in a. Data were presented as average of cooling index ± s.e.m. A one-way ANOVA was conducted and Tukey’s honest significant difference tests were used; *P < 0.05, **P < 0.005. n = number of flies. Individual P values are reported in the Source Data for this figure. Source data

Extended Data Figure 7 Sleep characteristics of wild-type and genetically manipulated flies under ramping temperature cycles and constant conditions after entrainment to ramping temperature cycles.

al, Sleep data analysis for Canton S and genetically modified flies under 18 °C to 25 °C temperature ramping cycles (see Methods). Daily total sleep time (a, e, i); total sleep time during heating (red) and cooling (blue) phases (b, f, j); sleep bout number during heating (red) and cooling (blue) phases (c, g, k); and sleep bout duration during heating (red) and cooling (blue) phases (d, h, l) of the genotypes indicated. Number of flies used in the analysis: ad, Canton S (n = 214), per01 (n = 96); eh, nocte+ (n = 40), nocte+ AL (n = 28), nocte1 (n = 49) and nocte1 AL (n = 39); il, Clk4.1m/+ (n = 17), UAS-TNT/+ (n = 23) and Clk4.1m>TNT (n = 100). Data are shown as population average ± s.e.m. n = number of flies. m, Statistical analysis. For determining statistical significance, ANOVA was performed across each individual group. Tukey’s honest significant difference tests were conducted in pairwise fashion with P values indicated on the graphs: **P < 0.005, *P < 0.05. Unpaired two-tailed Student’s t-tests were conducted for two-group comparison of the heating phase and the cooling phase within each individual genotype. The number of flies is shown in panels al. n, Averaged population sleep plots on day 1 of constant conditions after entrainment to ramping temperature cycles for wild-type Canton S (black, n = 198) and mutant per01 (red, n = 87) flies. Circadian time (CT) 0 is the start of the subjective heating phase and CT12 is the start of the subjective cooling phase, the times when the temperature transitions would have occurred had the temperature cycle continued. Dark lines indicate mean and shaded regions in the plots indicate s.e.m. s, Averaged population sleep plots on day 4 of constant temperature after entrainment for Canton S (black, n = 198) and per01 (red, n = 83) flies. Dark lines indicate mean and shaded regions in the plots indicate s.e.m. or, tw, Daily total sleep time, sleep times during subjective heating (red) and cooling (blue) phases, sleep bout number during subjective heating (red) and cooling (blue) phases, and sleep bout duration during subjective heating (red) and cooling (blue) phases of Canton S and per01 on day 1 (or) and day 4 (tw) of constant conditions. The number of flies in panels or is shown in n, and the number of flies in panels tw is shown in s. Data are presented as mean ± s.e.m. Canton S flies cycles under constant conditions display similar sleep patterns as observed during temperature cycles. By contrast, per01 flies do not display any differences in their sleep time or bout number or bout duration during the subjective heating phase and cooling phase, consistent with a lack of circadian timekeeping. n = number of flies. Statistical analysis was performed using unpaired two-tailed Student’s t-test. **P < 0.005, *P < 0.05. Individual P values are reported in the Source Data for this figure. Analysis of the sleep data revealed the following: 1. Wild-type flies sleep significantly more during the cooling phase than during the heating phase. 2. Wild-type flies have significantly more sleep bouts during the heating phase than during the cooling phase, indicating that sleep is more fragmented during the heating phase. 3. Wild-type flies have significantly longer sleep bout durations during the cooling phase compared to during the heating phase, indicating that sleep is more consolidated during the cooling phase. 4. Manipulation of chordotonal organ or aristae function does not produce changes in total sleep or sleep quality, only in the timing of sleep. 5. nocte1 mutant flies that lack aristae (nocte1 AL) fail to show significant differences in the amount of sleep during the heating and cooling phases. 6. Inhibition of a subset of DN1ps results in decreased total daily sleep time and specifically reduces sleep time during the heating phase. Source data

Extended Data Figure 8 Activity and sleep profiles of chordotonal organ mutants and flies lacking aristae.

a, b, Locomotor activity averaged over four days of a ramping temperature cycle (left) and averaged sleep profiles (right) for the following flies: a, nocte+ control flies (blue, n = 46) and nocte+ flies that lack aristae (nocte+ AL, red, n = 28); b, nocte1 mutants (blue, n = 56), and nocte1 mutants that lack aristae (nocte1 AL, red, n = 44). c, d, Locomotor activity profiles averaged over four days of a 22 °C to 28 °C (c) and a 18 °C to 28 °C (d) ramping temperature cycle for nocte+ flies (blue) and nocte1nocte1 mutants that lack aristae (nocte1 AL, red) (left); averaged sleep profiles (right). The number of flies in c and d are shown in e. e, Summary of locomotor activity rhythms under the two different temperature cycling conditions for the genotypes indicated. Data were presented as population average ± s.e.m. f, Locomotor activity averaged over four days of a ramping temperature cycle are shown on the left and averaged sleep profiles are shown on the right. The genotypes tested were F-gal4/+ (blue), UAS-nocteRNAi1/+ (green) and F-gal4> UAS-nocteRNAi1-AL (red), where AL refers to the aristae-less condition. The number of flies is shown in h. g, Anticipation of the onset of cooling in the genotypes indicated. F-gal4>UAS-nocteRNAi1-AL flies are largely arrhythmic (AR), so the anticipation index was not calculated (see Methods). Data are population average ± s.e.m. n values are shown in panel h. h, Summary of locomotor activity rhythms under constant conditions after entrainment. Data are mean ± s.e.m. i, Locomotor activity averaged over four days of a 18 °C to 25 °C ramping temperature cycle (left); averaged sleep profiles (right). nocte+ control flies are shown in blue and nocte1 mutants that lack aristae (nocte1 AL) are shown in red. The number of flies is shown j. j, Summary of locomotor activity rhythms under a ramping 18 °C to 25 °C temperature cycle and under constant conditions after entrainment for nocte+ flies and nocte1AL mutants. Data are mean ± s.e.m. k, l, Double-plotted actograms of a representative nocte+ fly (k) and nocte1 AL fly (l), which were entrained under temperature ramping cycles for 6 days and released into constant temperature (25 °C). The temperature gradient is shown at the bottom of the actogram. m, Locomotor activity averaged over four days of a 12 h:12 h light:dark cycle (left) and averaged sleep profiles (right) for nocte+ flies (blue) and nocte1 AL flies (red). The number of flies is shown in n. Lights are turned on at ZT0 and turned off at ZT12. n, Summary of locomotor activity rhythms under light:dark (LD) and subsequent constant dark:dark (DD) conditions for nocte+ flies and nocte1 AL mutants. Data are mean ± s.e.m. o, p, Double-plotted actograms of a representative nocte+ fly (o) and nocte1 AL fly (p), which were entrained to 12 h:12 h light:dark cycles for 6 days and released into constant darkness. In all activity and sleep plots, dark lines indicate mean and shaded regions indicate s.e.m. Black dashed lines in the plots represent temperature.

Extended Data Figure 9 Activity and sleep profiles of flies in which synaptic transmission is blocked in DN1p or DN2 clock neurons.

ac, Locomotor activity averaged over four days of a ramping temperature cycle (left) and averaged sleep profiles (right) for flies expressing tetanus toxin (TNT) in a subset of the DN1ps, DN1p>UAS-TNT experimental flies (red, n = 100), and DN1p>UAS-IMP-TNT controls expressing inactive TNT control (blue, n = 32) (a); glass60j mutants which lack all DN1ps (red, n = 49). and glass60j/+ heterozygotes (blue, n = 26) (b), and flies expressing TNT in the DN2s, DN2>TNT (red, n = 40) and DN2>IMP-TNT control (blue, n = 24) (c). Expression of TNT in DN2s did not affect the locomotor or sleep behaviour of flies under ramping temperature cycles under constant darkness. These results are consistent with previous findings that showed that DN2 synaptic output is required for behaviour synchronization in temperature cycles under constant light conditions, but not under constant dark conditions8. See Methods for detailed description of the genetic tools used. d, e, Averaged population locomotor activity on the day in which the temperature is transiently decreased or increased is shown for Clk4.1m>IMP-TNT control flies (n = 24) (d) and Clk4.1m>TNT flies (n = 20) (e). Flies were entrained to a 18 °C to 25 °C ramping temperature cycle for a week, and the temperature was decreased from 23 °C to 18 °C for an hour during the middle of the heating phase (blue bar) and increased from 22 °C to 25 °C for an hour in the middle of the cooling phase (red bar) on the eighth day of entrainment. Both the control and experimental flies show normal behavioural responses to unexpected changes in temperature, suggesting that blocking synaptic transmission from Clk4.1m expressing DN1ps does not have an effect on acute thermal responses. n = number of flies. In all the plots, dark lines indicate mean and shaded regions indicate s.e.m. Black dashed lines in the plots represent temperature.

Extended Data Figure 10 Activity profiles of flies in which clocks were sped up or slowed down in clock neurons.

a, b, Sleep plots and sleep slope plots for two Canton S flies (a) and two per01 flies (b). We identified ZT‘A’ (red dashed line) as the local maximum slope value closest to ZT12 (blue dashed line, the onset of cooling) such that sleep slopes at ZT‘A’ and for the next 2 h were non-negative. The difference between these two values (12 − A), which is quantified as the anticipation index, is indicated in each of the plots. See Methods for more details. c, d, To quantify the extent to which the major activity peak anticipates the onset of cooling, we determined the phase of the daily peak of activity closest to ZT12 (the onset of cooling) for individual flies. Representative raw activity profiles of an individual wild-type Canton S fly (c) and per01 mutant fly (d). Red lines represent a smoothed activity profile created by means of a Butterworth low-pass filter (see Methods). The phases of the activity peak closest to the onset of cooling at ZT12 are indicated in the plots. ZT0 corresponds to the start of the heating phase and ZT12 to the start of the cooling phase. el, Averaged population activity plots over four days of temperature cycles for the genotypes indicated. The number of flies is shown in m. In all plots, dark lines indicate mean and shaded regions indicate s.e.m. Dashed lines in the plots represent temperature. m, The average phases of the major activity peak closest to the onset of the cooling phase. 0 marks the start of the cooling phase, which corresponds to ZT12 in the locomotor activity plots shown above. The average phase values were calculated by taking the population average of the phase of the major activity peaks of individual flies. Error bars represent s.e.m. Sample sizes are reported to the right of the plot. Negative average phase values indicate that the major activity peak occurred before the onset of the cooling phase, and positive values indicate that the major peak occurred after cooling onset. ANOVAs were conducted to compare Clk4.1m>UAS-DBT(L) flies to Clk4.1m/+ and UAS-DBT(L)/+ controls (F2,95 = 26.1, P < 0.005); Clk4.1m>UAS-DBT(S) flies to Clk4.1m/+ and UAS-DBT(S)/+ controls (F2,76 = 6.5, P < 0.005); Clk856>UAS-DBT(L) flies to Clk856/+ and UAS-DBT(L)/+ controls (F2,78 = 18.4, P < 0.0005); and Clk856>UAS-DBT(S) flies to Clk856/+ and UAS-DBT(S)/+ controls (F2,77 = 25.1, P < 0.0005); Tukey’s tests are indicated. *P < 0.05, ***P < 0.005. Statistical analysis was performed using unpaired two-tailed Student’s t-tests. n, Population averages of the phases of the activity peak closest to the onset of cooling (indicated by the zero line). The number of flies used in the analysis is reported in Extended Data Fig. 6a. Error bars represent s.e.m. An ANOVA was performed for each group of genotypes and Tukey’s honest significant difference tests were conducted for pairwise comparisons. Statistical analysis was performed using unpaired two-tailed Student’s t-tests, **P < 0.005. Individual P values are reported in the Source Data for this figure. Source data

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Yadlapalli, S., Jiang, C., Bahle, A. et al. Circadian clock neurons constantly monitor environmental temperature to set sleep timing. Nature 555, 98–102 (2018). https://doi.org/10.1038/nature25740

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