Mechanical threat induces cardiac deceleration and locomotion

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 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. The time course of the experiment. An air puffs 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 20mL/min (gray), 50mL/min (sky blue), and 200mL/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 20mL/min (gray), 50mL/min (sky blue), and 200mL/min (dark blue), respectively. **p < 0.01, *p < 0.05, n.s.: p > 0.05, Wilcoxon’s signed rank test followed by Bonfferoni 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 20mL/min (gray), 50mL/min (sky blue), and 200mL/min (dark blue), respectively. ***p < 0.001, n.s.: p > 0.05, Wilcoxon’s signed rank test followed by Bonfferoni 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 20mL/min (gray), 50mL/min (sky blue), and 200mL/ min (dark blue), respectively. ***p < 0.001, n.s.: p > 0.05, Wilcoxon’s signed rank test followed by Bonfferoni correction.

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 red-shaded window. Lines and shaded areas represent means and SEM, respectively. N = 28. e. The HR averaged during 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℃ for 10 min and ramped up to 28℃ for 5min. Note it took approximately 17 s for the temperature to ramp from 21℃ to 28℃. j. Time course of the HR, shown as % change from average HR at 21℃. Temperature was rampted from 21℃ to 28℃ during red-shaded window. k. % Changes in the HR (28℃ vs 21℃) from the baseline (average over initial 60 s) averaged over 75s - 180s 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 HR during the 10 s baseline period preceding stimulation. N = 31, 29, 28, 35, 23, for +, WT, Dop1R1, Dop1R2, Dop2R, DopEcR,

Two pairs of dopaminergic neurons in the brain are paritally indispensable for cardiac deceleration

a. AsSchematic 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 HR during the 10 s baseline period preceding stimulation. N = 74, 22, 26, 21, 36, 20, 12, 25, 23, for +, TH-C, 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 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 um. Similar results were obtained across 3 independent samples. f. Brains of VT046828-AD ∩ R12D12-DBD>mCherry and R48A08-AD ∩ VT008692-DBD>mCherry (red) immunostained with anti-neuropil marker nc82 (blue). Scale bars: 25 um (inlet: 5 um). Similar results were obtained across 5 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 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.

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 5 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: 5μm. f. Time course of ΔF/F0. Puffs were applied at 1Hz for 10 times (over 10 s), as indicated by red-shaded boxes. N = 6 flies. g. ΔF/F0 averaged over 10s 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 10s, followed by 1s of light application, and 20s 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.

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, DA-WED>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, DA-WED>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 90s, followed by a 28ms light pulse, and 45s 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 breen-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. Model.