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 irregular electrical activity in the atria, which usually results in a fast and irregular ventricular rhythm⁴. Further, given the recent events of COVID-19, there has been an increased number of studies and evidence that show an increased appearance of arrhythmia in relation to COVID-19, mostly to sinus bradycardia⁵. On the opposite side of AF, sinus bradycardia presents a decreased and slower heart rhythm, with a heart rate lower than 60 beats per minute (bpm). This diagnosis requires an electrocardiogram (ECG) showing a normal sinus rhythm at a rate lower than 60 bpm.

The zebrafish, Danio rerio, has proven to be an ideal model for cardiovascular and drug development studies because of its homology to humans in both morphology, physiology, and genetics^6,7. Despite only having two discernible chambers compared to the four-chambered heart in humans, the zebrafish possesses a similar contractile structure with an analogous conduction system^8,9. Hence, the zebrafish model is suitable for researching cardiac arrhythmias and exploring the association between related genetic pathways and electrophysiological characteristics through ECG analysis. Regarding the sensor design for ECG recording in zebrafish, conventional needle electrodes are commonly used¹⁰. Other studies presented alternatives for conventional needles by designing a different type of needle made of different materials¹¹, these incorporated materials such as tungsten filament, stainless steel, and silver wire to assess the quality of the recorded data. Together with the portable ECG kit, they sought to establish a universal platform for both research and educational labs.

The needle system was also deployed in other studies^11,12,13,14 for carrying out research on biological and/or drug-induced effects, in which it showed encouraging outcomes. However, to obtain optimal signals, it was necessary to carefully insert the needles through the zebrafish's dermis. This invasive procedure can cause injury to the fish's heart due to the sharpness of the needles, thus possibly changing signal morphology¹³. Furthermore, accurately placing the electrode on the small heart without causing harm to ensure clear ECG capture presents a significant learning curve. Consequently, various substitute probe systems have been introduced, such as microelectrode arrays (MEA) and 3D-printed sensors. Both our team and other researchers have successfully utilized MEA for data acquisition, achieving data that offer a good signal-to-noise ratio (SNR) along with high spatial and temporal resolution^15,16,17. For example, we introduced an MEA that envelops the heart of the fish, allowing the capture of site-specific ECG signals^15,16. In contrast, another team developed a different kind of MEA based on a flexible printed circuit board (FPCB) crafted from polyimide film aimed at recording multiple electroencephalograms (EEGs) for epilepsy research¹⁸. While MEAs enable the recording of numerous signals, they are limited by the capacity to assess only one fish at a time due to the restricted number of electrode channels. In response to this limitation, we have recently showcased an advanced system capable of simultaneous ECG recordings from multiple fish¹⁹. This system employs two electrodes, each 125 µm in thickness and made of polyimide, with gold-sputtered electrodes positioned at the bottom of their housing to facilitate data acquisition. However, the inclusion of a water circulation pump introduced noise, affecting the signal-to-noise ratio (SNR) quality of the ECG signals. Additionally, the use of bulky and costly data collection tools, which required a wired connection to a computer, was necessitated. On the market, systems like iWORX offer improved portability through a compact amplifier, yet they face unresolved issues: (i) their recording time is too short (3-5 min) for experiments needing longer durations, such as those involving acute drug effects; (ii) the requirement for anesthetizing the animals, which can stress them and skew innate cardiac electrophysiological data; (iii) manual, sequential measurements hinder research with large numbers of fish; and (iv) the need for offline, labor-intensive ECG data processing. Importantly, no high-throughput systems incorporating microelectronics for analyzing mutant phenotypes have yet emerged. Therefore, the creation of high-throughput systems that enable extended ECG recording is crucial for uncovering links between arrhythmic phenotypes and mutant genotypes, identifying multiple arrhythmic phenotypes associated with single mutant genotypes, and testing the effectiveness of cardiac drugs using the zebrafish model. Consequently, we introduced, in 2022, the groundbreaking prototype of the Zebra II system. This novel system is equipped to perform extended ECG recordings from several fish at once. To achieve this, an in-house electronic system was crafted, utilizing the Internet of Things (IoT) technology for wireless data transmission and enabling data analysis through a mobile application. The inclusion of IoT technology not only bolstered the system's flexibility and portability but also facilitated remote collaborations for research on zebrafish models. In a prior study, we validated the system's efficacy through extensive testing, showcasing its ability to 1) acquire ECG data simultaneously from four fish; 2) perform continuous ECG recordings for up to 1 h, which significantly exceeds the capacity of existing systems that only capture a few minutes; 3) minimize the confounding effects of anesthesia by employing a 50% reduced concentration of Tricaine. Furthermore, the system demonstrated a strong capability for lengthy ECG recordings across various experiments, such as those involving sodium-induced sinoatrial (SA) block, temperature-induced heart rate changes, and drug-induced arrhythmias in both Tg(SCN5A-D1275N) mutant and wildtype fish under controlled experimental conditions. The system's adoption of multiple electrode channels for extended ECG recording further enables the execution of randomized controlled trials, allowing for two fish per experimental group to be studied under identical conditions²⁰. In this paper, we introduce the upgraded version of the system, featuring a 4-point electrode. This enhancement enables users to capture ECG readings from various locations on the fish, significantly reducing the chance of obtaining low-quality ECG signals. Moreover, this iteration builds upon and refines the majority of the functionalities outlined in previous research¹⁹. This evolved ECG system shows great promise in addressing existing limitations, thereby substantially facilitating the analysis of arrhythmic phenotypes and the screening of drugs in zebrafish. The innovative addition of the 4-point electrode was rigorously tested through a brief study on drug-induced arrhythmia in wild-type zebrafish, further validating its effectiveness.

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 tricaine powder and dissolve it in 97.9 mL of deionized (DI) water. Place a magnetic stirrer into the container and gradually mix the solution.
2. Adjust the pH by incrementally adding less than 17 mL of NaOH solution. Continue adding NaOH drops until the pH reaches a range of 6.8.-7.2.
3. Determine the final concentration of tricaine solution, accounting for the added NaOH necessary to adjust the pH and store.
   NOTE: Tricaine solution is light sensitive and should be stored in an amber-colored container at 4 °C when not in use.
4. Measure 258.13 mg of Amiodarone and dissolve it in 1 L of DI water. Insert a magnetic stirrer into the container and stir for a few minutes until the solution is completely dissolved and clear of any powder. Store the solution inside a refrigerator at 4°C when not in use.
   NOTE: Amiodarone must be prepared inside a fume hood and stored inside a refrigerator at 4 °C. Always wear the appropriate PPE.

2. Fish husbandry

1. Maintain the fish in individual tanks linked to a circulating water system, keeping the temperature between 28-29°C.
2. Ensure continuous monitoring of water quality and adhere to a 14 h/10 h day-night cycle.
3. Divide the 40 fish into four groups, each consisting of 10 fish, and house them in separate tanks.

3. Setting up the device

NOTE: The procedure outlined in step 3.2 must be performed for each chamber of the device (Figure 1). While each chamber can be set up differently, for consistency in the experiment, it is advisable to maintain uniform settings across all chambers.

1. Connect the device to a power source and initiate operation by pressing the Red Button located on the front panel.
2. Navigate through the device's touchscreen to choose the chamber to be configured.
   1. Adjust the low-pass filter setting to 0.1 Hz and the high-pass filter setting to 500 Hz. Adjust the gain setting to 10,000.
3. Lift the front lid to access the four chambers. Prior to starting the experiment, immerse each chamber's sponge (standard cellulose sponge) in Tricaine solution, then squeeze out any excess liquid.
4. Rotate the four-electrode array to ensure the sponge is accessible before introducing the fish.

4. Amiodarone treatment

NOTE: Utilize the water from the fish tanks (fish water) to prepare diluted solutions of Amiodarone and Tricaine. Steps 4.3 and 4.4 must be repeated for each group separated in step 2.3 for each solution prepared in step 4.1.

1. Follow the steps described below to dilute the Amiodarone stock solution.
   1. Dilute 8.75 mL of stock solution into 41.25 mL of fish water to achieve a 70 µM concentration.
   2. Dilute 12.5 mL of stock solution into 37.5 mL of fish water to achieve a 100 µM concentration.
   3. Dilute 25 mL of stock solution into 25 mL of fish water to achieve a 200 µM concentration.
2. Prepare the anesthesia solution by diluting 4 mL of Tricaine stock solution into 100 mL of fish water, resulting in a Tricaine concentration of 0.16 mg/mL
3. Submerge each fish individually in the prepared Amiodarone solution for 5 min. Use timers to accurately monitor the immersion duration.
4. Transfer the fish into Tricaine solution for 2-3 min for anesthesia. Ensure the treatment does not cause the death of the fish. If a fish exhibits abnormal behavior, such as losing color or a significant reduction in gill movement, remove it from the solution immediately and place it back into clean fish water.

5. ECG data collection

NOTE: Immediately follow these instructions after completing step 4.4. Repeat the process for each chamber before sealing the device. The chambers are designed to accommodate fish ranging from 4-10 mm in width and 25-50 mm in length.

1. Lift the fish out of the Tricaine solution and place it onto the sponge, ensuring its ventral side faces up.
2. Position the electrode array in a rhombus shape over the fish's chest area to capture ECG signals from four different points simultaneously (Figure 2A,B). Make sure to adjust each electrode carefully so every pin makes contact with the fish's skin.
   1. Rotate each electrode, lowering the pin until it makes contact with the fish's underside. If the pin is too high, turn the corresponding thumbscrew clockwise to lower it until it touches the fish.
   2. Perform the adjustment described above for the electrodes in all chambers.
3. Close the chambers' lid once all electrode arrays are correctly positioned over the fish in the operational chambers.
4. Mark each chamber for recording by selecting it on the touchscreen, then initiate the recording by clicking the Record button on-screen.
5. Click the Stop button after 2 min and then click on Save button from the prompt windows that pop ups on-screen after stopping. Save the information either on a disk or to the cloud, depending on specific research needs.

6. Troubleshooting the four-electrode array positioning system

NOTE: Occasionally, one or several electrodes might fail to capture a clear ECG signal, necessitating repositioning. Use the following steps to troubleshoot the electrode array when needed.

1. Press the Stop button on the device interface to halt the recording process and identify the channel or channels with inconsistent signals.
2. Lift the front lid for access to the electrode chambers. Identify which chamber(s) and specific electrodes are causing issues.
3. Turn the top thumbscrew clockwise to adjust the electrode's proximity to the fish. This action will lower the electrode closer to the fish.
4. Repeat step 6.3 as needed. If merely moving closer does not solve the issue, rotate the electrode to a different spot on the fish until a satisfactory signal is achieved.

7. Extracting and analyzing data

NOTE: All the basic data analysis presented in this paper uses the Wavelet technique²¹ to reduce various types of noise and standard deviation of normal sinus beats (SDNN) to calculate the beat-to-beat variance of HR in one-minute segments.

1. Once data has been saved to either the disk or the cloud, retrieve it for analysis or send it directly to a cloud server for further processing.
2. Navigate to the saved data by clicking the Browse button on the screen and select the data sets to use.
3. Select the Extract button on the options on-screen and attach a USB drive to the back part of the device to extract the data.
4. Follow the device prompts to save the selected data sets onto the external drive for subsequent retrieval and analysis.

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