Zebra II as A Novel System to Record Electrophysiological Signals in Zebrafish

Summary

The study introduces Zebra II, an advanced system for prolonged ECG acquisition and analysis in zebrafish. This system features an independent perfusion system and multiple-point electrodes for handling multiple fish in controlled environments. Acute effects of Amiodarone were assessed and analyzed with wild-type zebrafish.

Abstract

Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current study, the commercial version of the novel system, Zebra II, is presented. The protocol demonstrates electrocardiogram (ECG) acquisition and analysis from multiple zebrafish within controllable working environments. The device is composed of an external and independent perfusion system, a 4-point electrode, temperature sensors, and an embedded electronic system. In previous studies, the device prototype underwent validation against the established iWORX system through several tests, demonstrating similar data quality and ECG response to drug interventions. Following this, the study delved into examining the impact of anesthetic drugs and temperature fluctuations on zebrafish ECG, necessitating instant data evaluation. Thanks to the apparatus's capacity for consistent delivery of anesthetics and drugs, it was possible to extend ECG data collection up to 1 h, markedly longer than the 5 min duration supported by current systems. This paper introduces a pioneering, cloud-based, automated analysis utilizing data from four zebrafish, offering an efficient method for conducting combination experiments and significantly reducing time and effort. The system proved effective in capturing and analyzing ECG, especially in detecting drug-induced arrhythmias in wild-type zebrafish. Additionally, the capability to gather data across multiple channels facilitated the execution of randomized controlled trials with zebrafish models. The developed ECG system overcomes existing limitations, showing the potential to greatly expedite drug discovery and cardiovascular research involving zebrafish.

Introduction

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. According to the 2024 AHA Update Report, around 2552 people die every day of CVDs in the U.S., and the average direct and indirect cost of CVD was estimated at $378.0 billion in 2017-2018¹. Atrial fibrillation (AF) is one of the most common cardiac arrhythmias in the U.S.² and is associated with an unfavorable prognosis, increasing the risk of stroke and death in both CVDs and non-cardiovascular disease scenarios, leading to an increased risk of adverse outcomes³. This type of arrhythmia is distinguished by its rapid and irre....

Protocol

Adhering strictly to ethical guidelines ensures the well-being and appropriate handling of the zebrafish involved in our experiments. All experimental activities were conducted in accordance with the standards set forth by the Institutional Animal Care and Use Committees (IACUC). The experiments and findings detailed here were executed with the approval of IACUC under the authorized protocol AUP-21-066.

1. Preparation and storage of stock solutions

CAUTION: Prepare the tricaine stock solution in a fume hood, wearing appropriate personal protective equipment (PPE).

1. Measure 400 mg of tr....

Results

The ability to simultaneously record ECG signals from multiple fish significantly distinguishes this device, offering a considerable advantage in reducing the time required for drug or cardiac research on zebrafish or other similar species (Figure 1). Recording accurate ECG signals can be challenging due to the anatomical variations among fish, especially when it comes to electrode placement. Traditionally, obtaining a clear ECG signal involves positioning the active electrode over the heart.......

Discussion

The current steps were modified and improved according to this new device version from a previous study made by our team²⁰. Here, a labeled computer-aided design (CAD) of the device is also included.

As highlighted in the protocol's troubleshooting guide, the device necessitates the user to execute certain critical steps with diligence and precision, as these steps crucially affect the ECG signal's quality. Consistent with findings from our prior study^.......

Materials

Name	Company	Catalog Number	Comments
Amiodarone Hydrochloride	Sigma-Aldrich	PHR1164-1G	Amiodarone powder
Benchtop pH/mV Meter	Sper Scientific	10500308
Ethyle3-Aminobenzoate, Methanesulfonic Acid Salt, 98%, ACROS Organics	Fisher Scientific	AC118002500	Tricaine powder
Isotemp Hot Plate Stirrer	Fisher Scientific	SP88854200
Zebra II System	Sensoriis	N/A	Commercial Zebrafish ECG Recording System
Zebrafish ECG System	iWorx	ZS-200	Commercial Zebrafish ECG Recording System