Dopaminergic neurons linking threat processing to cardiac modulation and locomotor responses
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan
- International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, Japan
Figures
Figure 1 with 3 supplements
Mechanical threat induces cardiac deceleration and locomotion.
(a) Schematics of the paradigm. The fly is fixed on an air-supported ball. Left: the heart rate (HR) was videotaped by an IR-sensitive camera from the dorsal side through the cuticle (‘CAM1’). Of the four cardiac chambers, our system videotaped the chamber closest to the thorax called the conical chamber. We could easily distinguish diastole and systole in the captured video (blue dashed lines indicate the heart wall). Right: simultaneously, locomotion was videotaped from the right side of the fly (‘CAM2’). Blue dots and lines indicate automatically tracked body parts. (b) The time course of the experiment. An air puff of 0.5 s was applied once. The trial was repeated 10 times for each fly, the results of which were averaged. (c) An example M-mode of the heartbeat. An air puff was applied during the red-shaded time window. (d) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. The inlet shows the magnified view of the cardiac deceleration indicated by the box. (e) Change in HR, calculated for each fly as the % difference between the mean fraction during the 1 s following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. **p<0.01, *p<0.05, n.s.: p>0.05, Wilcoxon’s signed rank test followed by Bonferroni correction. (f) Time course of the walking fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. ***p<0.001, n.s.: p>0.05, Wilcoxon’s signed rank test followed by Bonferroni correction. (g) Change in walking fraction, calculated for each fly as the % difference between the mean fraction during the 10 s following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. ***p<0.001, n.s.: p>0.05, Wilcoxon’s signed rank test followed by Bonferroni correction. (h) Changes in HR and walking fraction jointly plotted for –1–3 s around the stimulus onset, for each puff strength. N=14, 17, 22 for 20, 50, and 200 mL/min, respectively.
Figure 1—figure supplement 1
heartbeat detection.
(a) The shown EIGHT consecutive frames capture an episode of ‘diastole ->systole ->diastole’ (‘Raw’, in which blue dotted lines indicate the outline of the conical chamber). Our software estimates the optical flow in these frames (‘Optical flow’, in which estimated directions are color-coded). Our software reveals a bout of contraction (frames 2–4), which is followed by a bout of relaxation (frames 5–8). Arrows indicate the optical flow direction for illustration purposes. (b) Plot comparing the HR calculated from manual count vs auto-detection, before and during puff application. Each line indicates identical flies. N=20. n.s.: p>0.05, two-tailed, paired t-test followed by Bonferroni correction. (c) Plot comparing the % change in HR (during vs pre-puff, 10 s each) calculated from manual count vs auto-detection. N=20. n.s.: p>0.05, two-tailed, paired t-test.
Figure 1—figure supplement 2
The effect of air puff on behavior and heartbeat.
(a) Time course of the HR (beats/s) without averaging over 10 trials. Results of different puff strengths are superimposed (gray: 20 mL/min, sky blue: 50 mL/min, dark blue: 200 mL/min; different fly groups were used for each puff strength). N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. (b) Time course of the HR averaged over 10 trials for each fly. Results of different puff strengths are superimposed (gray: 20 mL/min, sky blue: 50 mL/min, dark blue: 200 mL/min; different fly groups were used for each puff strength). N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. (c) Time course of the walking fraction (%) without averaging over 10 trials. Results of different puff strengths are superimposed (gray: 20 mL/min, sky blue: 50 mL/min, dark blue: 200 mL/min; different fly groups were used for each puff strength). N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. (d) Time course of walking fraction (%) averaged over 10 trials for each fly. Results of different puff strengths are superimposed (gray: 20 mL/min, sky blue: 50 mL/min, dark blue: 200 mL/min; different fly groups were used for each puff strength). N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. (e) Beat-to-beat interval (in seconds) plotted against the beat count around puff onset (gray vertical line). Dots and bars indicate mean and SEM, respectively. N=14, 17, 22 for 20, 50, and 200 mL/min, respectively. (f) Change in beat-to-beat interval, calculated for each fly as the % difference between the mean interval during three beats following stimulation onset and the mean interval during three beats preceding stimulation. N=14, 17, 22 for 20, 50, and 200 mL/min, respectively. ***p<0.001, **p<0.01, *p<0.05, Wilcoxon’s signed rank test followed by Bonferroni correction.
Figure 1—figure supplement 3
Somatosensory systems required for puff-induced cardiac deceleration.
(a) Drosophila possesses multiple mechanosensory organs throughout the body, including legs, halteres, wings, and the body trunk. To first distinguish which of these body parts may be indispensable for puff-induced cardiac deceleration, we surgically removed legs, halteres, wings, and tested puff-induced cardiac deceleration. Green indicates the heart. (b) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=37, 34, for intact flies, surgery flies, respectively. n.s.: p>0.05, Wilcoxon’s rank sum test. (c) Baseline HR. N=37, 34, for intact flies, surgery flies, respectively. *p<0.05, two-tailed t-test. (d) A schematic of the experiment. Different subsets of body hair somatosensors are silenced by expressing TNT using indicated driver lines. (Upper) split GAL4-based experiments. (Lower) GAL-based experiments. (e) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=36, 30, 27 for ‘empty’, ‘R38B08-AD R81E10-DBD’, ‘R38B08-AD VT043775-DBD’, respectively. *p<0.05, n.s.: p>0.05, Dunnett’s test. (f) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=43, 23, 25, 26, 20, 20 for ‘+’, ‘R74C07’, ‘Aes1N’, ‘Tes1N’, ‘iav’, ‘R48A07’, respectively. ***p<0.001, *p<0.05, n.s.: p>0.05, Dunnett’s test. (g) Baseline HR. N=36, 30, 27 for ‘empty’, ‘R38B08AD R81E10-DBD’, ‘R38B08-AD VT043775-DBD’, respectively. ***p<0.001, **p<0.01, Dunnett’s test. (h) Baseline HR. N=43, 23, 25, 26, 20, 20 for ‘+’, ‘R74C07’, ‘Aes1N’, ‘Tes1N’, ‘iav’, ‘R48A07’, respectively. ***p<0.001, Dunnett’s test. (i) Somatosensory neurons suggested to be partially indispensable for puff-induced cardiac deceleration. When multiple drivers labeled the given neurons, we considered such neurons as indispensable for cardiac deceleration only when those drivers produced consistent results (hence, e.g. external sensilla on the posterior abdomen were excluded based on Aes1N driver’s result, despite R74C07 driver’s result suggesting otherwise; similarly, sensilla on the eye compound labeled by R74C07 driver were excluded by ‘R38B08-AD VT043775-DBD’ split combination’s result.).
Figure 2 with 2 supplements
Dopamine system is responsible for puff-induced cardiac deceleration.
(a) Experimental setup. Hand enhancer drives the GFP expression in the cardiomyocytes (and in pericardiocytes). The fly was glued onto a slide glass, and the GFP signals were acquired through the intact cuticle. The air puffs were applied frontally to the fly’s head. (b) Example heartbeat. (Upper) Cartoon of the acquired image. Two rows of small cardiomyocytes and one pair of large pericardiocytes are visible. (Lower) Diastole and systole can be distinguished by the changing distance between two rows of cardiomyocytes (arrows). (c) The time course of the experiment. Air puffs of 0.5 s were applied at 1 Hz for 10 times (totalling 10 s). The trial was repeated twice for each fly, the results of which were averaged. (d) Time course of the HR. Air puffs are applied during the red-shaded window. Lines and shaded areas represent means and SEM, respectively. N=28. (e) The HR averaged during the puff application window (‘puff +’) vs 10 s before puff application (‘puff -’). Each line corresponds to each fly. N=28, ***p<0.001, two-tailed t-test. (f) Difference between puff + HR and puff- HR, calculated for each fly. N=28, ***p<0.001, two-tailed t-test. (g) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). Air puffs were applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. TNT expression is driven by indicated drivers. ‘+’ indicates driver-less control. Dashed line shows the lowest mean of ‘+’ control during puffs. N=55, 30, 29, 34, for +, TrH, Tdc2, TH, respectively. (h) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the mean HR during the 10 s baseline period preceding stimulation. N=55, 30, 29, 34, for +, TrH, Tdc2, TH, respectively. *p<0.05, n.s.: p>0.05, Dunn’s test. (i) Time course of the experiment. The heat plate beneath the slide glass warmed up the fly. The temperature of the slide glass is held at 21°C for 10 min and ramped up to 28°C for 5 min. Note it took approximately 17 s for the temperature to ramp from 21 to 28°C. (j) Time course of the HR, shown as % change from average HR at 21°C. Temperature was ramped from 21 to 28°C during the red-shaded window. (k) % Changes in the HR (28 vs 21°C) from the baseline (average over initial 60 s) averaged over 75–180 s window (shown as black bar in j.). N=33, 20, 14, 31, for +, TrH, Tdc2, TH, respectively. *p<0.05, n.s.: p>0.05, Dunn’s test. The upper dashed line shows the median of ‘+’ control. (l) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). Air puffs were applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=31, 29, 28, 35, 23, for +, WT, Dop1R1, Dop1R2, Dop2R, DopEcR, respectively. (m) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=31, 29, 28, 35, 23, for +, WT, Dop1R1, Dop1R2, Dop2R, DopEcR, respectively. **p<0.01, n.s.: p>0.05, Dunn’s test.
Figure 2—figure supplement 1
Heartbeat detection analysis.
(a) (Upper) example frames capturing the heart tube of a Hand-GFP fly. (Lower) Heat plot showing for each pixel the difference between consecutive frames. Pixels whose value at time t is larger than that at time (t-1) are shown with red; blue for the opposite. Double-headed arrows show the width of the heart for time t and (t-1) for illustrative purposes, and are not meant to be quantitatively precise. ‘SD(t)’ indicates the standard deviation of the y coordinates of red pixels, and ‘SD(t-1)’ of blue pixels. SD(t) is larger when the width of the heart is larger. Thus, ‘SD(t) - SD(t-1)’ indicates systole when <0, and diastole when >0. (b) Plot comparing the HR calculated from manual count vs auto-detection, before and during puff application. Each line indicates identical flies. N=27. **p<0.01, *p<0.05, two-tailed, paired t-test followed by Bonferroni correction. (c) Plot comparing the % change in HR (during vs pre-puff, 10 s each) calculated from manual count vs auto-detection. n.s.: p>0.05, two-tailed t-test. (d) Time course of the HR. A single puff was applied during red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=15. (e) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=15, *p<0.05, two-tailed t-test. (f) Time course of the HR. A train of 20 puffs was applied during red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=12. (g) Change in HR, calculated for each fly as the % difference between the mean HR during the 20 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=12, ***p<0.001, two-tailed t-test. (h) The baseline HR. N=13, 15 for male, female, respectively. n.s.: p>0.05, two-tailed t-test. (i) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=13, 15 for male, female, respectively. ***p<0.001, n.s.: p>0.05, two-tailed t-test. Sex-wise statistics were Bonferroni-corrected.
Figure 2—figure supplement 2
Roles of monoaminergic systems in puff-induced cardiac deceleration.
(a) Time courses of the HR in beats per s. A train of 10 puffs was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=55, 30, 29, 34, for ‘+’, TrH, Tdc2, TH, respectively. (b) The baseline HR in beats per s. N=55, 30, 29, 34, for ‘+’, TrH, Tdc2, TH, respectively. ***p<0.001, Dunnett’s test. (c) Time courses of the HR in beats per s. A train of 10 puffs was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=41, 22, 25, for ‘+>Kir’, ‘TrH>+’, ‘TdH >Kir’, respectively. (d) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=41, 22, 25, for ‘+>Kir’, ‘TrH>+’, ‘TdH >Kir’, respectively. ***p<0.001, *p<0.05, Dunnett’s test. (e) Time courses of the HR in beats per s. A train of 10 puffs was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=10, 14 for WT, TrH, respectively. (f) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=10, 14 for WT, TrH, respectively. *p<0.05, two-tailed t-test. (g) The baseline HR in beats per s. N=10, 14 for WT, TrH, respectively. n.s.: p>0.05, two-tailed t-test. (h) Time courses of the HR in beats per s. A train of 10 puffs was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=55, 29, 39, 35, 30, for WT, Dop1R1, Dop1R2, Dop2R, DopEcR, respectively. (i) The baseline HR in beats per s. N=55, 29, 39, 35, 30, for WT, Dop1R1, Dop1R2, Dop2R, DopEcR, respectively. ***p<0.001, n.s.: p>0.05, Dunn’s test.
Figure 3 with 1 supplement
Two pairs of dopaminergic neurons in the brain are partially indispensable for cardiac deceleration.
(a) Schematic of different DA-related GAL4 drivers. (b) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=74, 22, 26, 21, 36, 20, 12, 25, 23, for +, THC, TH-D, TH-F1, Vmat-R76F01, Vmat-R76F05, Ddc-R61H03, Ddc-R60F07, DAT-R55C10, respectively. **p<0.01, *p<0.05, n.s. (data shown in gray): p>0.05, Dunnett’s test. (c) A schematic of split-GAL4 strategy. (d) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=106, 27, 27, 26, 41, 32, 24, for driver-less control, R76F01 ∩ R76F02, R76F02 ∩ R76F05, R76F01 ∩ R76F02, R76F02 ∩ R76F07, R76F05 ∩ R60F07, R76F01 ∩ R60F07, R76F01 ∩ R76F05, respectively. *p<0.05, n.s.: p>0.05, Dunn’s test. (e) Brain of R60F07-AD ∩ R76F01-DBD>mCherry (red). An arrow indicates the soma of PPL1-SMPγ, while arrowheads indicate the soma of DA-WED. Scale bar: 25 µm. Similar results were obtained across three independent samples. (f) Brains of VT046828-AD ∩ R12D12-DBD>mCherry and R48A08-AD ∩ VT008692-DBD>mCherry (red) immunostained with antineuropil marker nc82 (blue). Scale bars: 25 µm (inlet: 5 µm). Similar results were obtained across five independent samples for each genotype. (g) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=51, 41, 36 for empty >TNT, PPL1-SMPγ>TNT, DA-WED>TNT, respectively. *p<0.05, n.s.: p>0.05, Dunnett’s test.
Figure 3—figure supplement 1
Silencing different subsets of dopaminergic neurons, DA-WED driver’s expression pattern, DA-WED silencing, and ple RNAi.
(a) The baseline HR in beats per s. N=74, 22, 26, 21, 36, 20, 12, 25, 23, 21, 33, for ‘+’, TH-C, TH-D, TH-F1, Vmat-R76F01, Vmat-R76F05, Ddc-R61H03, Ddc-R60F07, DAT-R55C10, DAT-B, Vmat-R76F02, respectively. ***p<0.05, n.s.: p>0.05, Dunn’s test. (b) The baseline HR in beats per s. N=106, 27, 27, 26, 41, 32, 24, for driver-less control, R76F01 ∩ R76F02, R76F02 ∩ R76F05, R76F01 ∩ R76F02, R76F02 ∩ R76F07, R76F05 ∩ R60F07, R76F01 ∩ R60F07, R76F01 ∩ R76F05, respectively. ***p<0.001, **p<0.01, n.s.: p>0.05, Dunn’s test. (c) The baseline HR in beats per s. N=51, 41, 36, for empty, PPL1-SMPγ, DA-WED, respectively. ***p<0.001, Dunn’s test. (d) Cleared tissue of a fly expressing mCD8GFP in DA-WED neurons R48A08-AD ∩ VT008692-DBD ∩ TH-FLP>mCD8::GFP (green). Autofluorescence of the tissue is visible as magenta. Arrowheads indicate DA-WED neuron’s soma. The bottom panels each show, from left to right, a foreleg, midleg, hindleg, and wing. Scale bar: 100 µm (inlet: 25 µm). (e) A brain and VNC of R48A08-AD ∩ VT008692-DBD ∩ TH-FLP>CsChrimson::mCherry (red) immunostained with anti-neuropil marker nc82 (blue). Scale bars: 25 µm (inlet: 5 um). Similar results were obtained across seven independent samples. Scale bar: 50 µm. (f) The baseline HR in beats per s. N=28, 27, for empty >Kir2.1, DA-WED>Kir2.1, respectively. n.s.: p>0.05, two-tailed t-test. (g) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). An air puff was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=40, 29, for EGFP-RNAi, ple-RNAi, respectively. (h) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=40, 29, for EGFP-RNAi, ple-RNAi, respectively. *p<0.05, two-tailed t-test. (i) The baseline HR. N=40, 29, for EGFP-RNAi, Ple-RNAi, respectively. *p<0.05, Wilcoxon’s signed rank test.
Figure 4 with 2 supplements
DA-WED neurons are responsible for puff-induced cardiac deceleration.
(a) The brain of R48A08-AD ∩ VT008692-DBD ∩ TH-FLP>mCD8::GFP (green) immunostained with anti-neuropil marker nc82 (blue). Scale bar: 30 um. Similar results were obtained across five independent samples. (b) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). Air puff was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=28, 27, for empty >Kir2.1, DA-WED>Kir2.1, respectively. (c) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=28, 27, for empty >Kir2.1, DA-WED>Kir2.1, respectively. *p<0.05, Two-tailed t-test. (d) Experimental setup. (e) Representative images of DA-WED neurons expressing GCaMP7s before and during puff application. Scale bar: 5 μm. (f) Time course of ΔF/F0. Puffs were applied at 1 Hz for 10 times (over 10 s), as indicated by red-shaded boxes. N=6 flies. (g) ΔF/F0 averaged over 10 s of puff application. When multiple neurons were recorded from the same fly (max 4 neurons per fly), results were averaged so that each dot represents one fly. N=6 flies. two-tailed t-test. **p<0.01. (h) Time course of the experiment. After 20 min of acclimation, the heartbeat of the fly expressing light-gated cation channel CsChrimson in DA-WED neurons, with (‘Ret +’) or without (‘Ret -’) being fed with the co-factor all-trans retinal (ATR) for 2 days prior to experiment, was recorded for 10 s, followed by 1 s of light application, and 20 s of recording. (i) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). Red light was shined during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=19, 22, for Ret-, Ret+, respectively. (j) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=13, 12, for Ret-, Ret+, respectively. *p<0.05, two-tailed t-test. (k) Model.
Figure 4—figure supplement 1
DA-WED neurons induce cardiac deceleration and protein feeding through distinct mechanisms.
(a) Time course of the experiment. Flies 2 days post eclosion were subjected to 8-day protein starvation protocol. Protein preference of the starved flies was then tested the next day. The preference test consisted of 3 hr food restriction, then 3 hr feeding on sucrose (supplemented with red dye) or yeast (supplemented with blue dye). The flies were then frozen and their abdominal color was inspected. (b) Protein preference test. Flies were transferred into a 35 mm petri dish, one half of which contained agarose gel with yeast + blue dye, while the other half contained sucrose + red dye. (c) Preference index was calculated as (# flies consuming yeast - # flies consuming sucrose) / (total # flies that fed). (d) Preference index. N=5, 5, 5, 4 batches (each with ~60 flies) for empty YD-, empty YD+, DA-WED YD-, DA-WED YD+, respectively. ***p<0.001, *p<0.05, two-tailed t-test. YD- vs YD +statistics were Bonferroni-corrected. (e) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=28, 27, for empty >Kir2.1, DA-WED>Kir2.1, respectively. *p<0.05, Two-tailed t-test. Figure 4c is shown here again for comparison with (d) (f) Preference index. N=5, 6, 6, 6 batches for WT YD-, WT YD+, Dop2R YD-, Dop2R YD+, respectively. *p<0.05, two-tailed t-test. YD- vs YD + statistics were Bonferroni-corrected. (g) Change in HR, calculated for each fly as the % difference between the mean HR during the 10 s stimulation period and the HR during the 10 s baseline period preceding stimulation. N=31, 35, for WT, Dop2R, respectively. Figure 2m is shown here again for comparison with (f) *p<0.05, Dunn’s test. (h) Preference index. N=4, 6, 4, 6 batches for empty YD-, empty YD+, FB-LAL YD-, FB-LAL YD+, respectively. ***p<0.001, *p<0.05, two-tailed t-test. YD- vs YD + statistics were Bonferroni-corrected. (i) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=27, 34, for empty >Kir2.1, FB-LAL>Kir2.1, respectively. n.s.: p>0.05, two-tailed t-test. (j) Preference index. N=12, 11 batches for YD-, YD+, respectively. ***p<0.001, two-tailed t-test. (k) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=14, 13, for YD-, YD+, respectively. n.s.: p>0.05, two-tailed t-test. (l) Baseline HR in beats per s. N=14, 13, for YD-, YD+, respectively. n.s.: p>0.05, Wilcoxon’s rank sum test. (m) Model.
Figure 4—figure supplement 2
Response of DA-WED neurons to air puffs.
(a) Rows depict the color-coded activity of a cell (z score). Red bar indicates the time window during which air puffs were applied. N=19 cells from 6 flies. (b) A cartoon showing medial and lateral DA-WED neurons. (c) Time course of ΔF/F0. Puffs were applied at 1 Hz for 10 times (over 10 s). (d) ΔF/F0 averaged over 10 s of puff application. When multiple neurons were recorded from the same fly (max 2 neurons per fly), results were averaged so that each dot represents one fly. Neurons for which medial/lateral location could not be distinguished are excluded from this analysis. N=3, 4 neurons for lateral and medial, respectively. Two-tailed t-test. n.s.: p>0.05.
Figure 5 with 2 supplements
Cardiac interoception possibly contributes to threat-induced locomotion.
(a) Time course of the walking fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). An air puff was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=28, 27, for empty >Kir2.1, DAWED >Kir2.1, respectively. (b) Change in walking fraction, calculated for each fly as the % difference between the mean fraction during the 10 s following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=28, 27, for empty >Kir2.1, DAWED >Kir2.1, respectively. **p<0.01, Two-tailed t-test. (c) Time course of the walking fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). Red light was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=13, 12, for ret -, +, respectively. (d) Change in walking fraction, calculated for each fly as the % difference between the mean fraction during the 10 s following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=13, 12, for ret -, +, respectively. **p<0.01, Two-tailed t-test. (e) A schematic of the strategy. Cardiomyocytes beat by oscillating between shrunk (systole) and relaxed (diastole) states. Diastole is driven by efflux of cation and resultant hyperpolarization. We expressed a light-gated anion channel GtACR1 in cardiomyocytes using Hand-GAL4, thus artificially driving hyperpolarization by shining light. (f) Time course of the experiment. After 20 min of acclimation, the heartbeat of the fly expressing GtACR1 in cardiomyocytes, with or without being fed with the co-factor all-trans retinal (ATR) for 2 days prior to experiment, was recorded for 90 s, followed by a 28ms light pulse, and 45 s of recording. The experiment was repeated twice for each fly. (g) An example M-mode during optogenetic cardiac deceleration. Green bar indicates when the light was shined. (h) Time course of the HR (% change from baseline defined as the average during the 10 s preceding puff onset). Green light was shined during the green-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=10, 11, for ret -, ret +, respectively. (i) Change in HR, calculated for each fly as the % difference between the mean HR during the 1 s following stimulation onset and the mean HR during the 10 s baseline period preceding stimulation. N=10, 11, for ret -, ret +, respectively. *p<0.05, two-tailed t-test. (j) Time course of the walking fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). Light was shined during the green-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=10, 11, for ret -, ret +, respectively. (k) Change in walking fraction, calculated for each fly as the % difference between the mean fraction during the 10 s following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=10, 11, for ret -, ret +, respectively. *p<0.05, two-tailed t-test. (l) Correlations between changes in walking fraction and HR. N=10, 11, for ret -, ret +, respectively. (m) Model. DA-WED neurons promote locomotion, through and/or bypassing interoception of the heartbeat (dashed arrows).
Figure 5—figure supplement 1
The effects of air puff, DA-WED manipulation, and optogenetic cardiac deceleration on grooming behavior.
(a) Time course of the grooming fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). An air puff was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. (b, c) Change in grooming fraction, calculated for each fly as the % difference between the mean fraction during the 3 s (b) or 10–20 s (c) following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=14, 17, 22 for 20 mL/min (gray), 50 mL/min (sky blue), and 200 mL/min (dark blue), respectively. ***p<0.001, **p<0.01, Wilcoxon’s signed rank test followed by Bonferroni correction. (d) Time course of the grooming fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). An air puff was applied during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=28, 27, for empty >Kir2.1, DA-WED>Kir2.1, respectively. (e, f) Change in grooming fraction, calculated for each fly as the % difference between the mean fraction during the 3 s (e) or 10–20 s (f) following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=28, 27, for empty >Kir2.1, DA-WED>Kir2.1, respectively. n.s.: p>0.05, Two-tailed t-test. (g) Time course of the grooming fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). Light was shined during the red-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=13, 12, for ret -, ret +, respectively. (h, i) Change in grooming fraction, calculated for each fly as the % difference between the mean fraction during the 3 s (h) or 10–20 s (i) following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=13, 12, for ret -, ret +, respectively. n.s.: p>0.05, Two-tailed t-test. (j) Time course of the grooming fraction (% change from baseline defined as the mean fraction during the 10 s preceding puff onset). Light was shined during the green-shaded time window. Lines and shaded areas represent means and SEM, respectively. N=10, 11, for ret -, ret +, respectively. (k, l) Change in grooming fraction, calculated for each fly as the % difference between the mean fraction during the 3 s (k) or 10–20 s (l) following stimulation onset and the mean fraction during the 10 s baseline period preceding stimulation. N=10, 11, for ret -, ret +, respectively. n.s.: p>0.05, two-tailed t-test. (m) Model. Air puff promotes locomotion while suppressing grooming. Mechanistically, air puff activates DA-WED neurons. DA-WED neurons drive not only cardiac deceleration but locomotion, potentially through a feedback from cardiac deceleration (dashed arrows). Such a DA-WED-based mechanism fails to influence grooming.
Figure 5—figure supplement 2
The effects of optogenetic cardiac deceleration on beat-to-beat interval.
(a) Correlations between changes in walking fraction and HR. N=14, 17, 22 for 20, 50, and 200 mL/min, respectively. Pearson’s correlation coefficient, followed by Bonferroni correction. (b) Beat-to-beat interval (in seconds) plotted against the beat count around puff onset (gray vertical line). Dots and bars indicate mean and SEM, respectively. N=11, 10 for ret -, +, respectively. (c) Change in beat-to-beat interval, calculated for each fly as the % difference between the mean interval during three beats following stimulation onset and the mean interval during three beats preceding stimulation. N=11, 10 for ret -, +, respectively. *p<0.05, Wilcoxon’s signed rank test. (d) Beat-to-beat interval (in seconds) plotted against the beat count around puff onset (gray vertical line). Dots and bars indicate mean and SEM, respectively. N=14, 17, 22 for 20, 50, and 200 mL/min, respectively. Figure 1—figure supplement 2e is shown here again for comparison. (e) Change in beat-to-beat interval, calculated for each fly as the % difference between the mean interval during three beats following stimulation onset and the mean interval during three beats preceding stimulation. N=14, 17, 22 for 20, 50, and 200 mL/min, respectively. ***p<0.001, **p<0.01, *p<0.05, Wilcoxon’s signed rank test followed by Bonferroni correction. Figure 1—figure supplement 2f is shown here again for comparison.
Videos
Video 1
An air puff induces cardiac deceleration and promotes locomotion.
A 50 mL/min puff is applied in the middle of the movie, as indicated by the red text that appears on the top left panel of the movie. The bottom left panel shows a behavioral recording, with automated body parts tracking (blue dots and lines) superimposed on the raw data. The top right panel shows the raw heartbeat recording, and the bottom right panel shows the pixel-wise, color-coded optical flow (color-to-direction mapping is shown on the top right of the panel). The playback speed is reduced to 0.25 of the original file.
Video 2
A train of 10 air puffs induces cardiac deceleration in a Hand >GFP fly.
The playback speed is reduced to 0.25 of the original file.
Video 3
Air puffs activate DA-WED neurons.
The left panel shows a schematic of the DA-WED neurons and the actual calcium imaging data (averaged over the total recording), capturing both DA-WED neurons in the right hemisphere. An arrow indicates the neuron shown on the right panel. The right panel shows GCaMP signals at time 0 on the top, and the time-lapse imaging from there during which air puffs were applied.
Video 4
Optogenetic activation of DA-WED neurons induces cardiac deceleration and promotes locomotion.
The playback speed is reduced to 0.25 of the original file.
Video 5
Optogenetic cardiac deceleration promotes locomotion.
The playback speed is reduced to 0.25 of the original file.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster, male) | Canton-S (CS) | Other | Gift from Dr. David Anderson (California Institute of Technology) | |
| Genetic reagent (D. melanogaster) | Hand-GFP | Other | Gift from Dr. Zhe Han (University of Maryland) | |
| Genetic reagent (D. melanogaster) | Hand-GAL4; R88D05-GAL4 | Bloomington Drosophila Stock Center | BDSC:48396; RRID:BDSC_48396 | |
| Genetic reagent (D. melanogaster) | TrH-GAL4 | Bloomington Drosophila Stock Center | BDSC:38388; RRID:BDSC_38388 | |
| Genetic reagent (D. melanogaster) | Tdc2-GAL4 | Bloomington Drosophila Stock Center | BDSC:9313; RRID:BDSC_9313 | |
| Genetic reagent (D. melanogaster) | TH-GAL4; ple-GAL4 | Bloomington Drosophila Stock Center | BDSC:8848; RRID:BDSC_8848 | |
| Genetic reagent (D. melanogaster) | TH-C-GAL4; TH-D-GAL4; TH-F1-GAL4 | Other | Gift from Dr. Mark N. Wu (Johns Hopkins University) | |
| Genetic reagent (D. melanogaster) | Vmat-R76F01-GAL4; Vmat-R76F05-GAL4; Ddc-R61H03-GAL4; Ddc-R60F07-GAL4; DAT-R55C10-GAL4 | Other | Gift from Dr. Mark N. Wu (Johns Hopkins University) | |
| Genetic reagent (D. melanogaster) | Vmat-R76F01-AD; Vmat-R76F05-AD; Ddc-R60F07-AD; Vmat-R76F01-DBD; Vmat-R76F05-DBD; Ddc-R60F07-DBD | Other | Gift from Dr. Mark N. Wu (Johns Hopkins University). Split-GAL4 hemidrivers. | |
| Genetic reagent (D. melanogaster) | TH-FLP | Other | Gift from Dr. Mark N. Wu (Johns Hopkins University) | |
| Genetic reagent (D. melanogaster) | UAS-FsF-mCD8GFP (UAS-FRT-stop-FRT-mCD8::GFP) | Other | Gift from Dr. Mark N. Wu (Johns Hopkins University) | |
| Genetic reagent (D. melanogaster) | Vmat-R76F02-GAL4; R76F02-AD; R76F02-DBD; Dat-B | Other | Gift from Dr. Mark N. Wu (Johns Hopkins University). Used in Figure 3b and d. | |
| Genetic reagent (D. melanogaster) | VT046828-AD | Bloomington Drosophila Stock Center | BDSC:73115; RRID:BDSC_73115 | |
| Genetic reagent (D. melanogaster) | R12D12-DBD | Bloomington Drosophila Stock Center | BDSC:69213; RRID:BDSC_69213 | |
| Genetic reagent (D. melanogaster) | R48A08-AD | Bloomington Drosophila Stock Center | BDSC:70707; RRID:BDSC_70707 | |
| Genetic reagent (D. melanogaster) | VT008692-DBD | Bloomington Drosophila Stock Center | BDSC:86909; RRID:BDSC_86909 | |
| Genetic reagent (D. melanogaster) | empty-AD; empty-DBD | Bloomington Drosophila Stock Center | BDSC:79603; RRID:BDSC_79603 | |
| Genetic reagent (D. melanogaster) | R38B08-AD | Bloomington Drosophila Stock Center | BDSC:71032; RRID:BDSC_71032 | |
| Genetic reagent (D. melanogaster) | R81E10-DBD | Bloomington Drosophila Stock Center | BDSC:68529; RRID:BDSC_68529 | |
| Genetic reagent (D. melanogaster) | VT043775-DBD | Bloomington Drosophila Stock Center | BDSC:73728; RRID:BDSC_73728 | |
| Genetic reagent (D. melanogaster) | R70G12-GAL4 | Bloomington Drosophila Stock Center | BDSC:39552; RRID:BDSC_39552 | |
| Genetic reagent (D. melanogaster) | R74C07-GAL4; Aes1N-GAL4 | Bloomington Drosophila Stock Center | BDSC:39847; RRID:BDSC_39847 | |
| Genetic reagent (D. melanogaster) | Tes1N-GAL4 | Kyoto Drosophila Stock Center | DGGR:103660; RRID:DGGR_103660 | |
| Genetic reagent (D. melanogaster) | iav-GAL4 | Bloomington Drosophila Stock Center | BDSC:52273; RRID:BDSC_52273 | |
| Genetic reagent (D. melanogaster) | R48A07-GAL4 | Bloomington Drosophila Stock Center | BDSC:50340; RRID:BDSC_50340 | |
| Genetic reagent (D. melanogaster) | UAS-TNT | Bloomington Drosophila Stock Center | BDSC:28838; RRID:BDSC_28838 | |
| Genetic reagent (D. melanogaster) | UAS-Kir2.1::EGFP | Bloomington Drosophila Stock Center | BDSC:6596; RRID:BDSC_6596 | |
| Genetic reagent (D. melanogaster) | UAS-FsF-Kir2.1 (UAS FRT-stop-FRT-Kir2.1) | Pther | Gift from Dr. Barry Dickson (The University of Queensland) | |
| Genetic reagent (D. melanogaster) | 20xUAS-CsChrimson::mVenus | Bloomington Drosophila Stock Center | BDSC:55135; RRID:BDSC_55135 | |
| Genetic reagent (D. melanogaster) | UAS-FsF-CsChrimson::mVenus (UAS-FRT-stop-FRT-CsChrimson::mVenus) | Other | Gift from Dr. Barry Dickson (The University of Queensland) | |
| Genetic reagent (D. melanogaster) | 20xUAS-CsChrimson::mCherry | Bloomington Drosophila Stock Center | BDSC:82181; RRID:BDSC_82181 | |
| Genetic reagent (D. melanogaster) | tub-FRT-GAL80-FRT (tub >GAL80>) | Bloomington Drosophila Stock Center | BDSC:38881; RRID:BDSC_38881 | |
| Genetic reagent (D. melanogaster) | UAS-GtACR1::tdTomato (III) | Other | Gift from Dr. Tetsuya Tabata (The University of Tokyo) | |
| Genetic reagent (D. melanogaster) | UAS-dTrpA1 | Bloomington Drosophila Stock Center | BDSC:26263; RRID:BDSC_26263 | |
| Genetic reagent (D. melanogaster) | UAS-mCD8::GFP | Bloomington Drosophila Stock Center | BDSC:32186; RRID:BDSC_32186 | |
| Genetic reagent (D. melanogaster) | UAS-mCherry | Bloomington Drosophila Stock Center | BDSC:52267; RRID:BDSC_52267 | |
| Genetic reagent (D. melanogaster) | UAS-RedStinger | Bloomington Drosophila Stock Center | BDSC:8547; RRID:BDSC_8547 | |
| Genetic reagent (D. melanogaster) | UAS-IVS-jGCaMP7s | Bloomington Drosophila Stock Center | BDSC:79032; RRID:BDSC_79032 | |
| Genetic reagent (D. melanogaster) | UAS-GFP RNAi (UAS-EGFP RNAi) | Bloomington Drosophila Stock Center | BDSC:41560; RRID:BDSC_41560 | |
| Genetic reagent (D. melanogaster) | UAS-Ple RNAi | Bloomington Drosophila Stock Center | BDSC:65875; RRID:BDSC_65875 | |
| Genetic reagent (D. melanogaster) | Dop1R1-attP; Dop1R2-attP; Dop2R-attP; DopEcR-attP | Other | Gift from Dr. Yi Rao (Peking University) | |
| Genetic reagent (D. melanogaster) | TrH-attP | Other | Gift from Dr. Yi Rao (Peking University) | |
| Antibody | Anti-Bruchpilot nc82 (mouse monoclonal) | Developmental Studies Hybridoma Bank | DSHB:nc82; RRID:AB_2314866 | IHC (1:10) |
| Antibody | Goat anti-mouse IgG Alexa Fluor 633 (goat polyclonal) | Molecular Probes / Thermo Fisher Scientific | Cat#A-21050; RRID:AB_2535718 | IHC (1:100) |
| Chemical compound, drug | All-trans retinal | Sigma-Aldrich | Cat#R2500-100MG | |
| chemical compound, drug | Brilliant blue FCF | Wako Pure Chemical Industries | CAS:3844-45-9 | |
| Chemical compound, drug | Sulforhodamine B | Sigma-Aldrich | CAS:3520-42-1 | |
| Other | Vectashield | Vector Laboratories | Cat#H-1000 | |
| Other | Bondic UV-curable adhesive | Bondic | Cat#BD-SKCJ | |
| Software, algorithm | SLEAP | Pereira et al., 2022 | RRID:SCR_021382 | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | |
| Software, algorithm | Python | Python Software Foundation | RRID:SCR_008394 | |
| Software, algorithm | R | R Core Team | RRID:SCR_001905 | |
| Software, algorithm | Custom analysis code (heartbeat detection, behavior classification, statistics) | This paper; https://github.com/mtsuji172/heart_behavior (copy archived at Tsuji, 2026) | v1.0.0 |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/111256/elife-111256-mdarchecklist1-v1.docx
-
Supplementary file 1
List of genotypes and sample sizes used in this study.
- https://cdn.elifesciences.org/articles/111256/elife-111256-supp1-v1.xlsx
-
Supplementary file 2
Statistics of data presented in this study.
- https://cdn.elifesciences.org/articles/111256/elife-111256-supp2-v1.xlsx
-
Source data 1
Numerical source data for all figure and figure-supplement panels (one worksheet per panel).
- https://cdn.elifesciences.org/articles/111256/elife-111256-data1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Dopaminergic neurons linking threat processing to cardiac modulation and locomotor responses
eLife 15:RP111256.
https://doi.org/10.7554/eLife.111256.3
