Illustration showing the system setup and the three-needle electrode placement during real-time adult zebrafish ECG recording. The zebrafish is anesthetized and immobilized on the immersed sponge. Two of the electrodes are positioned on micromanipulators. The electrode above the zebrafish’s heart is penetrated through the dermis with a micromanipulator. In this way, the penetration depth of the electrode probe can be measured by reading from the scale on the micromanipulator. ECG kit: The signals from the electrodes are amplified, filtered and converted to digital signals in this black box. (A) Scheme of ECG recording system. (B) Photograph of the experimental setup and positions of the recording probes. (C) Standard location of the pectoral recording probe in in vivo system as indicated by a cross in the field of view under a dissecting microscope.