Circadian neuron feedback controls the Drosophila sleep–activity profile

Journal name:
Nature
Volume:
536,
Pages:
292–297
Date published:
DOI:
doi:10.1038/nature19097
Received
Accepted
Published online

Abstract

Little is known about the ability of Drosophila circadian neurons to promote sleep. Here we show, using optogenetic manipulation and video recording, that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells that release glutamate to directly inhibit key pacemaker neurons. The pacemakers promote morning arousal by activating these DN1s, implying that a late-day feedback circuit drives midday siesta and night-time sleep. To investigate more plastic aspects of the sleep program, we used a calcium assay to monitor and compare the real-time activity of DN1 neurons in freely behaving males and females. Our results revealed that DN1 neurons were more active in males than in females, consistent with the finding that male flies sleep more during the day. DN1 activity is also enhanced by elevated temperature, consistent with the ability of higher temperatures to increase sleep. These new approaches indicate that DN1s have a major effect on the fly sleep–wake profile and integrate environmental information with the circadian molecular program.

At a glance

Figures

  1. Manipulation of DN1 activity affects the activity–sleep pattern.
    Figure 1: Manipulation of DN1 activity affects the activity–sleep pattern.

    a, Activity and sleep data for experimental (R18H11-GAL4/UAS-TNT) and control (R18H11-GAL4/UAS-inactive TNT) male flies. The recording paradigm used (video of 96-well plates or DAM tubes) is indicated by the cartoon. Left, activity data for control (left) and experimental (right) male flies in LD. White bars indicate daytime (ZT 0–12); black bars indicate night-time (ZT 12–24). Right, sleep traces for control (blue) and experimental groups (red). The genotypes are indicated below the panel. Shading corresponds to s.e.m. n = 14 GAL4>inactive TNT and 16 GAL4>TNT. bd, Stimulation and inhibition of DN1 firing modulate the E activity peak and sleep. b, Activity record of experimental flies. The pink boxes denote the red light (637 nm) stimulation window. c, Sleep traces from the baseline day (blue) and the red LED light stimulation day (red) of experimental flies. The pink bar represents the red light illumination (stripe bar upper, 10 HZ light pulse; solid bar lower, constant light). Shading corresponds to s.e.m. d, Quantification of sleep gained and lost between the baseline day (white background) and LED stimulation day (pink background) during ZT 7–12 (LED on times) in each group. n = 19 UAS-CsChrimson/+ and UAS-eNPHR3.0/+, n = 20 R18H11>CsChrimson and 21 R18H11>eNPHR3.0. Results with shading are mean ± s.e.m. **P < 0.001 by paired t-test.

  2. DN1s directly contact and reduce calcium levels in core pacemakers.
    Figure 2: DN1s directly contact and reduce calcium levels in core pacemakers.

    a, GRASP assays indicate contact between E cells, M cells and DN1s in the dorsal brain. Red signal indicates the large fragment of GFP (GFP1–10) expressed in the projections of E cells. The green signal shows the contact area between E cells and DN1s. The overlay is shown in the rightmost panel. Scale bar, 50 μm. Magnified images of the boxed area are shown in the middle. Scale bar, 20 μm. The GRASP signal (green) between DN1s (magenta) and M cells is shown in the lower row. b, c, DN1s inhibit calcium levels in the core pacemakers. b, Pseudo-coloured images of GCaMP6f fluorescence intensity from representative brains before and after ATP application. c, Mean GCaMP6f response traces are plotted. The genotypes for each group are labelled below. Solid line, ATP application. n = 8 for negative control, 7 for PDF cells and 11 for dorsal lateral neurons. Results in c are mean ± s.e.m. **P < 0.001 by unpaired two-tailed Student’s t-test.

  3. DN1s inhibit E cells via glutamate release to modulate the E peak and sleep.
    Figure 3: DN1s inhibit E cells via glutamate release to modulate the E peak and sleep.

    a, R18H11-driver labelled DN1s are glutamatergic. Anti-GFP (left) and anti-RFP (middle) staining was visualized in the dorsal brain of flies. Genotype is shown below. Scale bar, 20 μm. b, mGluRA mRNA levels cycle in E cells, peaking at midday. Two independent sets of 6 time points for mGluRA levels are plotted consecutively. c, Co-application of the mGluRA-specific antagonist LY 341495 with glutamate blocks the glutamate-induced inhibition. The 4 middle panels are representative GCaMP6f images of sLNvs during baseline, glutamate perfusion, wash out and glutamate plus LY 341495 perfusion (from left to right). The quantification of peak GCaMP6f changes is plotted in the two lower panels. n = 8 for left panel and n = 11 for right. **P < 0.001 by paired t-test. d, Perfusion of LY 341495 increases calcium levels within sLNvs. Mean GCaMP6f response traces are plotted. n = 11 for the experimental group and n = 7 for the negative control group. **P < 0.001 by unpaired two-tailed Student’s t-test. Results in panels are mean ± s.e.m. e, Reducing mGluRA within E cells impairs the DN1 activation effect on E peak and sleep. Left, the activity pattern of control and mGluRA RNAi flies at 21 °C and 27 °C. Right, quantification of daytime sleep changes from different groups. Box boundaries represent the first and third quartiles, and whiskers are 1.5 interquartile range. Genotypes are shown below. n = 31, 32, 32, 30 and 29 for genotypes in e, respectively. **P < 0.001 by one-way ANOVA with Tukey’s post-hoc test.

  4. DN1 neuronal activity is sexually dimorphic and can be activated by warm temperatures to enhance fly sleep.
    Figure 4: DN1 neuronal activity is sexually dimorphic and can be activated by warm temperatures to enhance fly sleep.

    a, Characterization of CaLexA–LUC in freely behaving animals. Left, LUC levels reflect neuronal activity in DN1s after CsChrimson stimulation (lower panel). The fold-change of luminescence was calculated as the ratio of the luminescence level after CsChrimson activation to the baseline luminescence level. The red-shaded box indicates the 10 min 627 nm light pulse. The genotypes of each line are shown below and n = 16 for each groups. Results with shading represents mean ± s.e.m. Right, CaLexA–LUC shows a marked male–female difference in DN1 activity. Averaged bioluminescence levels of 24 CLK4.1M-GAL4>CaLexA-LUC males (red) and females (grey) are plotted. Shaded background depicts dark periods. b, The real-time CaLexA–LUC assay reveals that warmer temperatures promote DN1 activity in the daytime. Black boxes indicate dark periods, white boxes indicate light periods. Flies were maintained at 21 °C and then transferred to 30 °C. Shading corresponds to s.e.m. Bioluminescence (arbitrary units); locomotor activity and daytime sleep profile are plotted (left) and quantified (right). n = 15 for each group; **P < 0.001 by paired t-test. c, Blocking DN1 output abolished warm-temperature-induced siesta in females. Sleep traces of control and experimental flies are shown on the left. Blue colour indicates data at 21 °C, and red colour indicates data at 30 °C. The box plot on right shows the sleep increase for the different groups. Box boundaries represent the first and third quartiles, whiskers are 1.5 interquartile range. n = 32 for each group and shading represent s.e.m. The genotype for each groups are labelled bellow. ** P < 0.001 by Kruskall–Wallis non-parametric one-way ANOVA with Dunn’s multiple comparisons test.

  5. Schematics of video recording and optogenetic strategies.
    Extended Data Fig. 1: Schematics of video recording and optogenetic strategies.

    a, Flies expressing CsChrimson were placed in 96-well plates and video recorded with a camera without an infrared filter (left). An 850 nm infrared back light provides illumination for recording in both light and dark periods. A set of 627 nm LEDs was carefully positioned and combined with a diffuser to ensure uniform irradiation for stimulation. The voltage and pulse frequency were controlled by an Arduino UNO board as described in Methods. Representative data from a video recording of male and female activity (right) in light:dark are shown. b, Sleep data of two control genotypes in 96-well plate mode for 6 days. Results with error bars are mean ± s.e.m. n = 15–16 for each group.

  6. The R18H11 driver labels a subgroup of CLK4.1M-defined DN1s.
    Extended Data Fig. 2: The R18H11 driver labels a subgroup of CLK4.1M-defined DN1s.

    Confocal stack of images showing antibody staining for GFP (left) and RFP (middle) and the overlay (right) in the dorsal brain of R18H11-LexA/LexAop-mCD8::GFP;Clk4.1M-GAL4/UAS-rCD2::RFP flies. Scale bar, 20 μm.

  7. Blocking neurotransmitter output of DN1s affects sleep parameters but not DD rhythmicity.
    Extended Data Fig. 3: Blocking neurotransmitter output of DN1s affects sleep parameters but not DD rhythmicity.

    af, Total sleep, maximum sleep bout duration and mean sleep bout duration of control groups (R18H11-GAL4/+, UAS-TNT/+) and the experimental group (R18H11-GAL4/UAS-TNT) have significant differences. Results with error bars are mean ± s.e.m. n = 32 for each group. One-way ANOVA was performed to detect significant genotype effects for total sleep (P < 0.0001), daytime sleep (P < 0.0001), night-time sleep (P < 0.0001) (a, b), maximum bout duration (P = 0.00288), maximum daytime bout duration (P < 0.0001), maximum night-time bout duration (P = 0.000388) (d), mean bout duration (P < 0.0001), mean daytime bout duration (P < 0.0001), mean night-time bout duration (P < 0.0001) (e). Asterisks denote significant differences from parental controls in Tukey’s post-hoc test (P < 0.01). g, Neurotransmitter release from DN1s is not required for DD rhythmicity. Locomotor behaviour of control (Clk4.1M-GAL4/UAS-Tet) and experimental (Clk4.1M-GAL4/UAS-TNT) male flies was monitored for 6 days in DD. Both control (left) and experimental (right) flies maintained strong rhythmicity. Note that the experimental group showed much less daytime sleep and higher activity level (dashed red arrow-right panel) than the control group (solid red arrow-left panel). n = 32 for each group.

  8. Co-expression of CRY-GAL80 or TNT blocks the sleep-promoting effect of DN1 activation.
    Extended Data Fig. 4: Co-expression of CRY-GAL80 or TNT blocks the sleep-promoting effect of DN1 activation.

    a, R18H11-GAL4/UAS-CsChrimson (left) and R18H11-GAL4, CRY-GAL80/UAS-CsChrimson (right) brains were dissected and stained with anti-GFP (green). Scale bar, 100 μm. b, Comparison of total sleep in the baseline day (blue) to total sleep during a 24 h LED stimulation day (red) for each genotype. n = 32 for R18H11-GAL4/UAS-CsChrimson and n = 24 for the other groups. Results with error bars are mean ± s.e.m. **P < 0.001 by post-hoc Bonferonni multiple comparisons. Two-way ANOVA was performed to detect a significant LED stimulation effect (P = 0.00227674), a genotype effect (P < 0.0001) and interaction (P < 0.0001).

  9. Arousal threshold is affected by manipulation of DN1 activity.
    Extended Data Fig. 5: Arousal threshold is affected by manipulation of DN1 activity.

    a, Mechanical stimulation setup for measuring arousal threshold. The set-up is illustrated on the left. A 96-well plate is loaded onto the device and a small push–pull solenoid is positioned on the side of the plate. The solenoid can be programed to tap the plate at different frequencies and times. A web camera monitors fly movement in the wells, and the video is analysed with Fiji ImageJ software to track flies. An example image is shown on the right. b, Activation of DN1s increases arousal threshold. The left panel shows representative trajectories (5 min traces) of experimental and control flies after a strong (10 tap) stimulus at ZT6 when the LED is on. The right panel shows the percentages of flies for the indicated genotypes that transitioned from immobility to an active state in response to the stimulus. The pink background indicates LED stimulation. n = 24 for R18H11-GAL4/UAS-CsChrimson and R18H11-GAL4/UAS-TNT groups, n = 16 for parental control groups. Results with error bars are mean ± s.e.m. One-way ANOVA was performed to detect significant genotype effects for arousal levels of R18H11>CsChrimson LED group (P < 0.0001) and R18H11>TNT group (P < 0.0001). Asterisks denote significant differences from parental controls in Tukey’s post-hoc test (P < 0.01).

  10. eNPHR3.0 blocks DN1 neuronal activity and decreases the siesta.
    Extended Data Fig. 6: eNPHR3.0 blocks DN1 neuronal activity and decreases the siesta.

    a, The luminescence traces from the CaLexA–LUC sensor reflect neuronal activity after LED-induced eNPHR3.0 inhibition from ZT 2.5 to ZT 9.5. The mean LUC activity level (arbitrary units) from control and experimental groups is quantified on the right. Results with shading are mean ± s.e.m. Box boundaries represent the first and third quartiles, whiskers are 1.5 interquartile range. n = 24 for each group. b, Sleep from a baseline day and from a LED stimulation day of R18H11>UAS-eNPHR3.0, UAS-eNPHR3.0/+ and R18H11/+ flies. Pink background represents LED stimulation. n = 16 for each group. Results with error bars are mean ± s.e.m.

  11. The dendritic region of E cells overlaps with the presynaptic region of DN1s.
    Extended Data Fig. 7: The dendritic region of E cells overlaps with the presynaptic region of DN1s.

    Clk4.1M-GAL4>UAS-synaptotagmin-GFP brains were dissected and stained with anti-GFP antibodies to identify the DN1 presynaptic regions (green; left panel). To identify the dendritic regions of E cells and M cells, DvPdf-GAL4>UAS-Denmark brains were dissected and stained with anti-DsRed antibodies (red; middle panel). These patterns were aligned and overlaid (merged panel on the right).

  12. Glutamate reduces calcium levels in PDF neurons and hyperpolarizes their membrane potential.
    Extended Data Fig. 8: Glutamate reduces calcium levels in PDF neurons and hyperpolarizes their membrane potential.

    a, Quantification of peak GCaMP6f changes shown in Fig. 2c. Average maximum changes for LNvs and dorsal lateral neurons are shown. n = 8 for control group, 7 for PDF cells and 11 for dorsal lateral neurons. **P < 0.001 by unpaired two-tailed Student’s t-test and results with error bars are mean ± s.e.m. b, Normalized calcium traces in different circadian neuron subgroups imaged concurrently in the same representative brain. c, d, 5 mM glutamate was applied to exposed dissected fly brains (Pdf-GAL4>GCaMP6f, (c) and Pdf-GAL4>Arclight (d)) and induced a calcium decrease and hyperpolarized these core pacemakers (ΔF/F represents the evoked fluorescence change from baseline). The red solid line indicates time of glutamate application. The dashed line indicates time of vehicle application. Panels show representative data of 6 brains.

  13. Decreasing DN1 VGLUT levels blocks the E peak reduction caused by DN1 firing.
    Extended Data Fig. 9: Decreasing DN1 VGLUT levels blocks the E peak reduction caused by DN1 firing.

    Reducing VGLUT activity within the DN1s decreases the DN1-activation effect (left). The locomotor activity patterns of UAS-dTrpA1/+; CLK4.1M-GAL4/+ female flies (upper panel) and UAS-dTrpA1/+; CLK4.1M-GAL4/UAS-VGlut RNAi female flies (lower panel) at 21 °C and 27 °C are shown. The colour of the bars indicates either daytime (white) or night-time (black). Evening activity index was calculated as described in the Methods for the indicated genotypes (right). White and black bars represent data from low and high temperature respectively. **P < 0.001 by unpaired two-tailed Student’s t-test. n = 24 for each group. Results with error bars are mean ± s.e.m.

  14. Reducing mGluRA expression in pacemaker neurons reduces the inhibitory effect of glutamate as well as the siesta.
    Extended Data Fig. 10: Reducing mGluRA expression in pacemaker neurons reduces the inhibitory effect of glutamate as well as the siesta.

    a, The peak decrease of GCaMP6f in circadian cells after applying glutamate to control and mGluRA knockdown flies. The genotypes are shown above the bars. n = 6 UAS-Dcr2; Clk856-GAL4, UAS-GCaMP6f and n = 8–9 UAS-Dcr2; Clk856-GAL4, UAS-GCaMP6f; UAS-mGluRA RNAi groups. *P < 0.05 by unpaired t-test. b, Comparison of total sleep, daytime siesta and night-time sleep in different genotypes. n = 32 for each group. *P < 0.05 by one-way ANOVA with Tukey’s post-hoc test. Results with error bars are mean ± s.e.m (a, b). c, A temporally constrained negative feedback core pacemaker–DN1 circuit regulates the fly activity–sleep pattern. Early in the day, M pacemaker neurons activate the DN1s via the PDF neuropeptide, and DN1s release DH31 to enhance morning arousal. Later in the day, glutamate release from DN1s inhibits M cells and E cells, promotes the siesta, decreases the evening activity peak and initiates night-time sleep. A cycling mRNA that encodes inhibitory glutamate receptors in pacemaker cells may help direct this inhibition to the late day. This feedback circadian circuit shapes the bimodal locomotor activity peak and sleep–wake cycles under normal conditions. The higher daily neuronal activity in male DN1s compared to female DN1s promotes the sexually dimorphic activity/sleep pattern. DN1s also integrate environmental information such as temperature to promote sleep plasticity.

Videos

  1. Live calcium imaging of circadian neuron responses to DN1 activation
    Video 1: Live calcium imaging of circadian neuron responses to DN1 activation
    The calcium responses of clock neurons expressing GCaMP6f to ATP/P2X2-activation of DN1s. The genotype of flies used in the experiment is Clk856-GAL4/UAS-GCaMP6f; Clk4.1M-LexA/LexAop-P2X2. This video is accelerated 15X. ATP was added from 30 S (2S in the video).
  2. R18H11-GAL4-labeled DN1 arbors co-localize with VGLUT
    Video 2: R18H11-GAL4-labeled DN1 arbors co-localize with VGLUT
    Confocal stack of antibody staining of GFP and VGLUT (red) in the dorsal brain of R18H11-GAL4>UAS-mCD8::GFP flies. Most R18H11-GAL4-labeled DN1s are glutamatergic as VGLUT is highly enriched in their projections.

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Author information

Affiliations

  1. Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts 02454, USA

    • Fang Guo,
    • Hyung Jae Jung,
    • Katharine C. Abruzzi,
    • Weifei Luo &
    • Michael Rosbash
  2. Department of Biology, National Center for Behavioral Genomics and Volen National Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA

    • Fang Guo,
    • Junwei Yu,
    • Hyung Jae Jung,
    • Katharine C. Abruzzi,
    • Weifei Luo,
    • Leslie C. Griffith &
    • Michael Rosbash

Contributions

F.G. and M.R. conceived and designed the experiments. F.G. performed behavioural experiments. F.G. and J.Y. performed immunocytochemistry and imaging experiments. F.G. and H.J.J. setup the recording system. K.C.A. and W.L. performed and quantified the mRNA profiling data. F.G. and J.Y. analysed data. F.G., L.G. and M.R. prepared the figures and wrote the paper, with feedback from all authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Reviewer Information

Nature thanks A. Sehgal and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Schematics of video recording and optogenetic strategies. (240 KB)

    a, Flies expressing CsChrimson were placed in 96-well plates and video recorded with a camera without an infrared filter (left). An 850 nm infrared back light provides illumination for recording in both light and dark periods. A set of 627 nm LEDs was carefully positioned and combined with a diffuser to ensure uniform irradiation for stimulation. The voltage and pulse frequency were controlled by an Arduino UNO board as described in Methods. Representative data from a video recording of male and female activity (right) in light:dark are shown. b, Sleep data of two control genotypes in 96-well plate mode for 6 days. Results with error bars are mean ± s.e.m. n = 15–16 for each group.

  2. Extended Data Figure 2: The R18H11 driver labels a subgroup of CLK4.1M-defined DN1s. (263 KB)

    Confocal stack of images showing antibody staining for GFP (left) and RFP (middle) and the overlay (right) in the dorsal brain of R18H11-LexA/LexAop-mCD8::GFP;Clk4.1M-GAL4/UAS-rCD2::RFP flies. Scale bar, 20 μm.

  3. Extended Data Figure 3: Blocking neurotransmitter output of DN1s affects sleep parameters but not DD rhythmicity. (414 KB)

    af, Total sleep, maximum sleep bout duration and mean sleep bout duration of control groups (R18H11-GAL4/+, UAS-TNT/+) and the experimental group (R18H11-GAL4/UAS-TNT) have significant differences. Results with error bars are mean ± s.e.m. n = 32 for each group. One-way ANOVA was performed to detect significant genotype effects for total sleep (P < 0.0001), daytime sleep (P < 0.0001), night-time sleep (P < 0.0001) (a, b), maximum bout duration (P = 0.00288), maximum daytime bout duration (P < 0.0001), maximum night-time bout duration (P = 0.000388) (d), mean bout duration (P < 0.0001), mean daytime bout duration (P < 0.0001), mean night-time bout duration (P < 0.0001) (e). Asterisks denote significant differences from parental controls in Tukey’s post-hoc test (P < 0.01). g, Neurotransmitter release from DN1s is not required for DD rhythmicity. Locomotor behaviour of control (Clk4.1M-GAL4/UAS-Tet) and experimental (Clk4.1M-GAL4/UAS-TNT) male flies was monitored for 6 days in DD. Both control (left) and experimental (right) flies maintained strong rhythmicity. Note that the experimental group showed much less daytime sleep and higher activity level (dashed red arrow-right panel) than the control group (solid red arrow-left panel). n = 32 for each group.

  4. Extended Data Figure 4: Co-expression of CRY-GAL80 or TNT blocks the sleep-promoting effect of DN1 activation. (147 KB)

    a, R18H11-GAL4/UAS-CsChrimson (left) and R18H11-GAL4, CRY-GAL80/UAS-CsChrimson (right) brains were dissected and stained with anti-GFP (green). Scale bar, 100 μm. b, Comparison of total sleep in the baseline day (blue) to total sleep during a 24 h LED stimulation day (red) for each genotype. n = 32 for R18H11-GAL4/UAS-CsChrimson and n = 24 for the other groups. Results with error bars are mean ± s.e.m. **P < 0.001 by post-hoc Bonferonni multiple comparisons. Two-way ANOVA was performed to detect a significant LED stimulation effect (P = 0.00227674), a genotype effect (P < 0.0001) and interaction (P < 0.0001).

  5. Extended Data Figure 5: Arousal threshold is affected by manipulation of DN1 activity. (347 KB)

    a, Mechanical stimulation setup for measuring arousal threshold. The set-up is illustrated on the left. A 96-well plate is loaded onto the device and a small push–pull solenoid is positioned on the side of the plate. The solenoid can be programed to tap the plate at different frequencies and times. A web camera monitors fly movement in the wells, and the video is analysed with Fiji ImageJ software to track flies. An example image is shown on the right. b, Activation of DN1s increases arousal threshold. The left panel shows representative trajectories (5 min traces) of experimental and control flies after a strong (10 tap) stimulus at ZT6 when the LED is on. The right panel shows the percentages of flies for the indicated genotypes that transitioned from immobility to an active state in response to the stimulus. The pink background indicates LED stimulation. n = 24 for R18H11-GAL4/UAS-CsChrimson and R18H11-GAL4/UAS-TNT groups, n = 16 for parental control groups. Results with error bars are mean ± s.e.m. One-way ANOVA was performed to detect significant genotype effects for arousal levels of R18H11>CsChrimson LED group (P < 0.0001) and R18H11>TNT group (P < 0.0001). Asterisks denote significant differences from parental controls in Tukey’s post-hoc test (P < 0.01).

  6. Extended Data Figure 6: eNPHR3.0 blocks DN1 neuronal activity and decreases the siesta. (176 KB)

    a, The luminescence traces from the CaLexA–LUC sensor reflect neuronal activity after LED-induced eNPHR3.0 inhibition from ZT 2.5 to ZT 9.5. The mean LUC activity level (arbitrary units) from control and experimental groups is quantified on the right. Results with shading are mean ± s.e.m. Box boundaries represent the first and third quartiles, whiskers are 1.5 interquartile range. n = 24 for each group. b, Sleep from a baseline day and from a LED stimulation day of R18H11>UAS-eNPHR3.0, UAS-eNPHR3.0/+ and R18H11/+ flies. Pink background represents LED stimulation. n = 16 for each group. Results with error bars are mean ± s.e.m.

  7. Extended Data Figure 7: The dendritic region of E cells overlaps with the presynaptic region of DN1s. (233 KB)

    Clk4.1M-GAL4>UAS-synaptotagmin-GFP brains were dissected and stained with anti-GFP antibodies to identify the DN1 presynaptic regions (green; left panel). To identify the dendritic regions of E cells and M cells, DvPdf-GAL4>UAS-Denmark brains were dissected and stained with anti-DsRed antibodies (red; middle panel). These patterns were aligned and overlaid (merged panel on the right).

  8. Extended Data Figure 8: Glutamate reduces calcium levels in PDF neurons and hyperpolarizes their membrane potential. (498 KB)

    a, Quantification of peak GCaMP6f changes shown in Fig. 2c. Average maximum changes for LNvs and dorsal lateral neurons are shown. n = 8 for control group, 7 for PDF cells and 11 for dorsal lateral neurons. **P < 0.001 by unpaired two-tailed Student’s t-test and results with error bars are mean ± s.e.m. b, Normalized calcium traces in different circadian neuron subgroups imaged concurrently in the same representative brain. c, d, 5 mM glutamate was applied to exposed dissected fly brains (Pdf-GAL4>GCaMP6f, (c) and Pdf-GAL4>Arclight (d)) and induced a calcium decrease and hyperpolarized these core pacemakers (ΔF/F represents the evoked fluorescence change from baseline). The red solid line indicates time of glutamate application. The dashed line indicates time of vehicle application. Panels show representative data of 6 brains.

  9. Extended Data Figure 9: Decreasing DN1 VGLUT levels blocks the E peak reduction caused by DN1 firing. (137 KB)

    Reducing VGLUT activity within the DN1s decreases the DN1-activation effect (left). The locomotor activity patterns of UAS-dTrpA1/+; CLK4.1M-GAL4/+ female flies (upper panel) and UAS-dTrpA1/+; CLK4.1M-GAL4/UAS-VGlut RNAi female flies (lower panel) at 21 °C and 27 °C are shown. The colour of the bars indicates either daytime (white) or night-time (black). Evening activity index was calculated as described in the Methods for the indicated genotypes (right). White and black bars represent data from low and high temperature respectively. **P < 0.001 by unpaired two-tailed Student’s t-test. n = 24 for each group. Results with error bars are mean ± s.e.m.

  10. Extended Data Figure 10: Reducing mGluRA expression in pacemaker neurons reduces the inhibitory effect of glutamate as well as the siesta. (134 KB)

    a, The peak decrease of GCaMP6f in circadian cells after applying glutamate to control and mGluRA knockdown flies. The genotypes are shown above the bars. n = 6 UAS-Dcr2; Clk856-GAL4, UAS-GCaMP6f and n = 8–9 UAS-Dcr2; Clk856-GAL4, UAS-GCaMP6f; UAS-mGluRA RNAi groups. *P < 0.05 by unpaired t-test. b, Comparison of total sleep, daytime siesta and night-time sleep in different genotypes. n = 32 for each group. *P < 0.05 by one-way ANOVA with Tukey’s post-hoc test. Results with error bars are mean ± s.e.m (a, b). c, A temporally constrained negative feedback core pacemaker–DN1 circuit regulates the fly activity–sleep pattern. Early in the day, M pacemaker neurons activate the DN1s via the PDF neuropeptide, and DN1s release DH31 to enhance morning arousal. Later in the day, glutamate release from DN1s inhibits M cells and E cells, promotes the siesta, decreases the evening activity peak and initiates night-time sleep. A cycling mRNA that encodes inhibitory glutamate receptors in pacemaker cells may help direct this inhibition to the late day. This feedback circadian circuit shapes the bimodal locomotor activity peak and sleep–wake cycles under normal conditions. The higher daily neuronal activity in male DN1s compared to female DN1s promotes the sexually dimorphic activity/sleep pattern. DN1s also integrate environmental information such as temperature to promote sleep plasticity.

Supplementary information

Video

  1. Video 1: Live calcium imaging of circadian neuron responses to DN1 activation (19.69 MB, Download)
    The calcium responses of clock neurons expressing GCaMP6f to ATP/P2X2-activation of DN1s. The genotype of flies used in the experiment is Clk856-GAL4/UAS-GCaMP6f; Clk4.1M-LexA/LexAop-P2X2. This video is accelerated 15X. ATP was added from 30 S (2S in the video).
  2. Video 2: R18H11-GAL4-labeled DN1 arbors co-localize with VGLUT (15 MB, Download)
    Confocal stack of antibody staining of GFP and VGLUT (red) in the dorsal brain of R18H11-GAL4>UAS-mCD8::GFP flies. Most R18H11-GAL4-labeled DN1s are glutamatergic as VGLUT is highly enriched in their projections.

Additional data