Fig. 1: Drosophila circadian neurons respond to changes in ambient temperature.

a Schematic of the Drosophila circadian neuronal circuit. Left panel is the EM reconstruction of circadian neurons and right panel is a Clk856-GAL4>GCaMP6s brain stained with GFP antibodies. b Schematic of the two-photon imaging system with precise temperature control and fly-on-ball setups. c, f Representative pseudocolor images of calcium responses of Clk856-GAL4>UAS-GCaMP6s flies before and after cooling (c) and heating (f). white arrow indicates DN1as. d, g Heat map of the temporal calcium activities of circadian neurons to cooling (24–18 °C, d) and heating (22–30 °C, g). The temperature change was labeled under the panel. e, h The representative GCaMP traces (ΔF/F₀ ± SEM) of DN1as in response to cooling (blue, n = 16, e) and heating (red, n = 16, h). e related to (d). h related to (g). i Expression pattern of the DN1as driven by DN1a-spl in the brain revealed by anti-tdTomato (cyan) and anti-NC82 (gray). Scale bar, 20 μm. j The representative GCaMP traces (ΔF/F ± SEM) of DN1as in response to cooling (left) and heating (right) with steps of different amplitude (red: ΔT = 2 °C; blue: ΔT = 4 °C; green: ΔT = 6 °C; purple: ΔT = 8 °C; orange: ΔT = 10 °C, n = 8). k Quantification of the relative fold change of calcium activities of DN1as in (j). Data are presented as mean in ΔF/F₀ ± SEM in the histogram; Statistical analysis was conducted using One-Way ANOVA followed by Tukey post-test for multiple comparisons. The letters A, B, C, D, E, F and G above the histograms denote significantly different means within each of the two groups, p < 0.05. Specific p values corresponding to this figure are reported in the Source Data. n = 8.