Actogram j

Zebrafish (Danio rerio) larvae (3.5 dpf) were placed in each well of a 96 deep-well plate (96 Well Clear Assay Plate Pyramid Flat Bottom); 800 μL E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂· 2H₂O, 0.33 mM MgSO₄· 7H₂O, 10 mM HEPES, pH 7.2) containing the compound (10 μg/mL) was added to each well. The plate was set on DanioVision (Noldus) and the behavior of the fish was tracked under constant darkness with a CCD camera with infrared light. The amount of activity (i.e., distance moved) per minute was calculated using the image processing function of EthoVision XT11 (Noldus). The free-running period and phase were calculated using Actogram J and MultiCycle (Actimetrics), respectively¹⁹ . All animal studies were carried out in accordance with ARRIVE guidelines and all methods were in compliant with relevant guidelines and regulations and were approved by the Animal Experiment Committee of Nagoya University.

The locomotor activity of mice was monitored throughout the experiment individually in cages equipped with infrared movement detectors positioned centrally above the top of each cage, and the activity was detected using a circadian activity monitoring system (Dr. H.M. Cooper, INSERM, France) as required. The activity was recorded every minute, and double-plotted actograms and Chi-square periodograms [37 (link)] were generated to visualize the data with Chi-square statistics set at P < 0.0001; significant periodogram peaks within the circadian range of SCNx mice were below 10% of an average Ctrl peak (Fig. S1). The parameters of circadian rhythmicity were analyzed using the ClockLab toolbox (Actimetrics, USA) and Fiji ImageJ plugin ActogramJ [38 (link)].