Name: Zebra II as A Novel System to Record Electrophysiological Signals in Zebrafish (Video) | JoVE Journal
Uploaded: 2024-08-16T14:30:00.000+00:00
Duration: 6 min 15 s
Description: Discover the Zebra II system for prolonged ECG acquisition in zebrafish using multichannel electrodes. This method enables up to one hour of cardiac recording for drug screening and cardiac gene studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We are grateful for the support from the NIH SBIR Phase II #R44OD024874 to H.C. and M.P.H.L. The authors would also like to acknowledge the financial support provided by the Consejo Nacional de Humanidades, Ciencias y Tecnologias (CONAHCYT) in Mexico through the fellowship titled Beca CONAHCYT Para Estudios de Doctorado en el Extranjero.

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).
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C when not in use.</li>
	<li>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&#176;C when not in use. 
	NOTE: Amiodarone must be prepared inside a fume hood and stored inside a refrigerator at 4 &#176;C. Always wear the appropriate PPE.</li>
</ol>
2. Fish husbandry
<ol>
	<li>Maintain the fish in individual tanks linked to a circulating water system, keeping the temperature between 28-29&#176;C.</li>
	<li>Ensure continuous monitoring of water quality and adhere to a 14 h/10 h day-night cycle.</li>
	<li>Divide the 40 fish into four groups, each consisting of 10 fish, and house them in separate tanks.</li>
</ol>
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.
<ol>
	<li>Connect the device to a power source and initiate operation by pressing the Red Button located on the front panel.</li>
	<li>Navigate through the device&#39;s touchscreen to choose the chamber to be configured.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Lift the front lid to access the four chambers. Prior to starting the experiment, immerse each chamber&#39;s sponge (standard cellulose sponge) in Tricaine solution, then squeeze out any excess liquid.</li>
	<li>Rotate the four-electrode array to ensure the sponge is accessible before introducing the fish.</li>
</ol>
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.
<ol>
	<li>Follow the steps described below to dilute the Amiodarone stock solution.
	<ol>
		<li>Dilute 8.75 mL of stock solution into 41.25 mL of fish water to achieve a 70 &#181;M concentration.</li>
		<li>Dilute 12.5 mL of stock solution into 37.5 mL of fish water to achieve a 100 &#181;M concentration.</li>
		<li>Dilute 25 mL of stock solution into 25 mL of fish water to achieve a 200 &#181;M concentration.</li>
	</ol>
	</li>
	<li>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</li>
	<li>Submerge each fish individually in the prepared Amiodarone solution for 5 min. Use timers to accurately monitor the immersion duration.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Lift the fish out of the Tricaine solution and place it onto the sponge, ensuring its ventral side faces up.</li>
	<li>Position the electrode array in a rhombus shape over the fish&#39;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&#39;s skin.
	<ol>
		<li>Rotate each electrode, lowering the pin until it makes contact with the fish&#39;s underside. If the pin is too high, turn the corresponding thumbscrew clockwise to lower it until it touches the fish.</li>
		<li>Perform the adjustment described above for the electrodes in all chambers.</li>
	</ol>
	</li>
	<li>Close the chambers&#39; lid once all electrode arrays are correctly positioned over the fish in the operational chambers.</li>
	<li>Mark each chamber for recording by selecting it on the touchscreen, then initiate the recording by clicking the Record button on-screen.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Press the Stop button on the device interface to halt the recording process and identify the channel or channels with inconsistent signals.</li>
	<li>Lift the front lid for access to the electrode chambers. Identify which chamber(s) and specific electrodes are causing issues.</li>
	<li>Turn the top thumbscrew clockwise to adjust the electrode&#39;s proximity to the fish. This action will lower the electrode closer to the fish.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Navigate to the saved data by clicking the Browse button on the screen and select the data sets to use.</li>
	<li>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.</li>
	<li>Follow the device prompts to save the selected data sets onto the external drive for subsequent retrieval and analysis.</li>
</ol>

Zebra II: Advanced ECG System for Multichannel Zebrafish Cardiovascular Studies

<ol><li>Tsao, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+disease+and+stroke+statistics-2022+update%3A+A+report+from+the+American+heart+association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Heart disease and stroke statistics-2022 update: A report from the American heart association.</a> Circulation. 145 (8), e153-e639 (2022).</li>
<li>Zulkifly, H., Lip, G. Y. H., Lane, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+of+atrial+fibrillation%5BTitle%5D)+AND+%22Int+J+Clin+Pract%22%5BJournal%5D)">Epidemiology of atrial fibrillation.</a> Int J Clin Pract. 72 (3), e13070(2018).</li>
<li>Ferreira, C., Providência, R., Ferreira, M. J., Gonçalves, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atrial+fibrillation+and+non-cardiovascular+diseases%3A+A+systematic+review%5BTitle%5D)+AND+%22Arq+Bras+Cardiol%22%5BJournal%5D)">Atrial fibrillation and non-cardiovascular diseases: A systematic review.</a> Arq Bras Cardiol. 105 (5), 519-526 (2015).</li>
<li>Hu, Z., Ding, L., Yao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atrial+fibrillation%3A+mechanism+and+clinical+management%5BTitle%5D)+AND+%22Chin+Med+J%22%5BJournal%5D)">Atrial fibrillation: mechanism and clinical management.</a> Chin Med J. 136 (22), 2668-2676 (2023).</li>
<li>Babapoor-Farrokhran, S., Rasekhi, R. T., Gill, D., Babapoor, S., Amanullah, A. Arrhythmia in COVID-19. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBabapoor-Farrokhran%2C+S+%22SN+Comp+Clin+Med%22">SN Comp Clin Med</a>. 2 (9), 1430-1435 (2020).</li>
<li>Arnaout, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+model+for+human+long+QT+syndrome%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Zebrafish model for human long QT syndrome.</a> Proc Natl Acad Sci. 104 (27), 11316-11321 (2007).</li>
<li>Koopman, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+grime+mutant+uncovers+an+evolutionarily+conserved+role+for+Tmem161b+in+the+control+of+cardiac+rhythm%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">The zebrafish grime mutant uncovers an evolutionarily conserved role for Tmem161b in the control of cardiac rhythm.</a> Proc Natl Acad Sci. 118 (9), e2018220118(2021).</li>
<li>Genge, C. E., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGenge%2C+CE+%22Reviews+of+Physiology%2C+Biochemistry+and+Pharmacology%22">Reviews of Physiology, Biochemistry and Pharmacology</a>. , Springer International Publishing. (2016).</li>
<li>Nemtsas, P., Wettwer, E., Christ, T., Weidinger, G., Ravens, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+zebrafish+heart+as+a+model+for+human+heart%3F+An+electrophysiological+study%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Adult zebrafish heart as a model for human heart? An electrophysiological study.</a> J Mol Cell Cardiol. 48 (1), 161-171 (2010).</li>
<li>Sun, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-electrocardiograms+to+study+post-ventricular+amputation+of+Zebrafish+heart%5BTitle%5D)+AND+%22Ann+Biomed+Engg%22%5BJournal%5D)">Micro-electrocardiograms to study post-ventricular amputation of Zebrafish heart.</a> Ann Biomed Engg. 37 (5), 890-901 (2009).</li>
<li>Lin, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+rapid+and+economic+in+vivo+electrocardiogram+platform+for+cardiovascular+drug+assay+and+electrophysiology+research+in+adult+zebrafish%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Development of a rapid and economic in vivo electrocardiogram platform for cardiovascular drug assay and electrophysiology research in adult zebrafish.</a> Sci Rep. 8 (1), 15986(2018).</li>
<li>Chaudhari, G. H., Chennubhotla, K. S., Chatti, K., Kulkarni, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+adult+zebrafish+ECG+method+for+assessment+of+drug-induced+QTc+prolongation%5BTitle%5D)+AND+%22J+Pharmacol+Toxicol+Methods%22%5BJournal%5D)">Optimization of the adult zebrafish ECG method for assessment of drug-induced QTc prolongation.</a> J Pharmacol Toxicol Methods. 67 (2), 115-120 (2013).</li>
<li>Liu, C. C., Li, L., Lam, Y. W., Siu, C. W., Cheng, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improvement+of+surface+ECG+recording+in+adult+zebrafish+reveals+that+the+value+of+this+model+exceeds+our+expectation%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Improvement of surface ECG recording in adult zebrafish reveals that the value of this model exceeds our expectation.</a> Sci Rep. 6, 25073(2016).</li>
<li>Stoyek, M. R., Rog-Zielinska, E. A., Quinn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-associated+changes+in+electrical+function+of+the+zebrafish+heart%5BTitle%5D)+AND+%22Prog+Biophys+Mol+Biol%22%5BJournal%5D)">Age-associated changes in electrical function of the zebrafish heart.</a> Prog Biophys Mol Biol. 138, 91-104 (2018).</li>
<li>Cao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wearable+multi-channel+microelectrode+membranes+for+elucidating+electrophysiological+phenotypes+of+injured+myocardium%5BTitle%5D)+AND+%22Integ+Biol%22%5BJournal%5D)">Wearable multi-channel microelectrode membranes for elucidating electrophysiological phenotypes of injured myocardium.</a> Integ Biol. 6 (8), 789-795 (2014).</li>
<li>Lenning, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+monitoring+and+analysis+of+zebrafish+electrocardiogram+with+anomaly+detection%5BTitle%5D)+AND+%22Sensors%22%5BJournal%5D)">Real-time monitoring and analysis of zebrafish electrocardiogram with anomaly detection.</a> Sensors. 18 (1), 61(2018).</li>
<li>Yu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flexible+microelectrode+arrays+to+interface+epicardial+electrical+signals+with+intracardial+calcium+transients+in+zebrafish+hearts%5BTitle%5D)+AND+%22Biomed+Microdev%22%5BJournal%5D)">Flexible microelectrode arrays to interface epicardial electrical signals with intracardial calcium transients in zebrafish hearts.</a> Biomed Microdev. 14 (2), 357-366 (2012).</li>
<li>Cho, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+an+animal+model+in+epilepsy+studies+with+multichannel+EEG+recordings%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Zebrafish as an animal model in epilepsy studies with multichannel EEG recordings.</a> Sci Rep. 7 (1), 3099(2017).</li>
<li>Continuous electrocardiogram monitoring in Zebrafish with prolonged mild anesthesia. Le, T., et al. 42nd Ann Int Conf IEEE Engg Med Biol Soc, , 2610-2613 (2020).</li>
<li>Le, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+wireless+ECG+system+for+prolonged+monitoring+of+multiple+zebrafish+for+heart+disease+and+drug+screening+studies%5BTitle%5D)+AND+%22Biosensors+Bioelectron%22%5BJournal%5D)">A novel wireless ECG system for prolonged monitoring of multiple zebrafish for heart disease and drug screening studies.</a> Biosensors Bioelectron. 197, 113808(2022).</li>
<li>Kouzehgar, K., Eslamian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+2+-+Application+of+experimental+data+and+soft+computing+techniques+in+determining+the+outflow+and+breach+characteristics+in+embankments+and+landslide+dams%5BTitle%5D)+AND+%22Handbook+Hydroinfo%22%5BJournal%5D)">Chapter 2 - Application of experimental data and soft computing techniques in determining the outflow and breach characteristics in embankments and landslide dams.</a> Handbook Hydroinfo. 3, 11-31 (2023).</li>
<li>Milan, D. J., Jones, I. L., Ellinor, P. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+recording+of+adult+zebrafish+electrocardiogram+and+assessment+of+drug-induced+QT+prolongation%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation.</a> Am J Physiol Heart Circ Physiol. 291 (1), H269-H273 (2006).</li>
</ol>

Hung Cao and Michael Lau are the founders of Sensoriis, Inc. Sensoriis's system Zebra II was developed under NIH support, SBIR Phase II grant #R44OD024874. The University of California, Irvine, has reviewed and approved Hung Cao's relationship with Sensoriis, Inc. in accordance with its conflict-of-interest policies. The other authors declare no conflict of interest.

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 study20, the positioning of the electrodes plays a significant role in acquiring high-quality and clear ECG readings from zebrafish samples. Due to this method expanding the number of working electrodes from one to four and introducing additional degrees of freedom, it demands supplementary troubleshooting steps and refined electrode placement over the fish's ventral area for optimal results.
The advantages and novel features of the device lie in extended measurement for multi-step experiments, limited to hour-long studies, high throughput screening with multiple zebrafish (four simultaneous studies), highly controllable experiment setup, additional electrophysiology recordings per sample (up to four) and automated cloud-based analytics and data storing. From this, we would like to emphasize that the major issue being addressed is the time needed to perform drug screening and cardiac studies in zebrafish, as traditional methods require a one-by-one screening and multiple trials to gather enough information22. This also offers a new way of using multi-channel arrays to record ECG in a simpler way compared to those that use microelectrode arrays (MEA) or flexible printed circuit boards (FPCB)18 which required extra steps in the fabrication of the mentioned electrodes plus the procedure needed to attach them to the fish compared to the proposed method that also relies on surface contact but in a less invasive way.
The limitations of the system are not different from those presented in traditional methods19,20 but are less intensive as a multi-channel array is used to decrease the number of recording trials needed for the study. These limitations rely on the electrode positioning, as it is recommended to follow the proposed positioning presented in Figure 2B. It also needs to be clear that the user cannot expect to have perfect recordings during the first trial on the four channels as the anatomical differences of fish and the pre-processing of the sample (des-scaling, open-chest procedures, etc.) will directly affect the quality and success of the tests.

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 genetics6,7. 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 system8,9. 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 studies11,12,13,14 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&#39;s dermis. This invasive procedure can cause injury to the fish&#39;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 resolution15,16,17. For example, we introduced an MEA that envelops the heart of the fish, allowing the capture of site-specific ECG signals15,16. 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 &#181;m 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&#39;s flexibility and portability but also facilitated remote collaborations for research on zebrafish models. In a prior study, we validated the system&#39;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&#39;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.

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

zebra ii as a novel system to record electrophysiological signals in zebrafish

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

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&#39;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.

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 to circumvent capturing mechanical beats or motion artifacts, with the reference electrode placed towards the lower abdomen. However, pinpointing the heart's precise location can be difficult due to its depth within the tissue or the fish's uneven surface. Sometimes, electrodes must be maneuvered with micromanipulators for accurate placement, which may require multiple attempts before capturing a quality signal.
To overcome these challenges, the system introduces an innovative approach by using a configuration of four electrodes instead of conventional single-working electrodes. Each electrode operates independently, allowing adjustments in three dimensions-up and down (heave), tilting forward and backward (pitch), and rotating around the vertical axis (roll). This flexibility proves particularly useful when working with fish of various shapes, as it enables precise electrode positioning to either capture the optimal ECG signal or explore how ECG readings vary with different contact points on the fish.
This feature not only enhances the efficiency of cardiac and drug studies but also broadens the possibilities for ECG and even ECG recording techniques. Given that these methods have yet to be standardized and are often customized to accommodate the specific conditions of the experimental subject, this device opens new avenues for research, offering more adaptable and reliable data collection methods (Figure 2).
One of the distinctive features of this system is its ability to evaluate up to four different drugs concurrently on four separate fish or to investigate the effects of up to four different doses of a single drug across four distinct fish. The experimental setup can range from a brief acute exposure to longer sessions spanning 1 h, involving variable drug dosages or changes in environmental conditions such as temperature and anesthesia. In this study, we conducted a brief experiment to demonstrate the method's efficacy, particularly highlighting the functionality of the four-electrode array. We tested three varying doses of Amiodarone, a class-III anti-arrhythmic drug known for inducting bradycardia and prolonging the QT interval in zebrafish, over a 5 min exposure period, similar to a previous study20 using the first prototype of this device.
Consistent with prior research, the fish showed both a prolonged QTc interval and a reduction in heart rate compared to the untreated control group. Despite the brief exposure time, which is shorter than in many comparable studies that extend to 1 h, we were able to observe significant alterations in the ECG signals and cardiac parameters (Figure 3).
How Amiodarone treatment elicited observable modifications in the ECG, which escalated with an increase in dosage, is illustrated in Figure 3A,B. Notably, the QTc interval extended with each successive dosage increase relative to the control group, which exhibited a QTc interval of 330 ms. Following the treated groups, the QTcs intervals were measured at 365 ms for 70 µM of Amiodarone, 480 ms for 100 µM, and 546 ms for 200 µM. Correspondingly, a noteworthy decrease in heart rate was observed in reaction to the varied Amiodarone dosages and the prolonged QTcs intervals. Heart rate dropped significantly from an average of 120 ±5 BPM in the control group to 105 ±10 BPM at 70 µM of Amiodarone, 90 ± 5 BPM at 100 µM, and 84 ±5 BPM at 200 µM (Figure 3C,D).
Based on these findings, we can conclude that the newly proposed technique demonstrates comparable quality to previous studies17,18, employing a two-electrode approach (one working electrode and one reference electrode). We successfully gathered data from four working electrodes, selecting the optimal one for analysis to then report the anticipated cardiac parameters. The criteria for selection align with those of earlier methods, emphasizing the need to distinctly visualize PQRST complex and low baseline noise in the ECG signal. This enables the calculation of QT and QTc intervals and facilitates the identification of clear differences. Also, the methodology underwent further validation through testing by three additional researchers from our group who had not participated in the development of the current device. They first implemented the preliminary methodology suggested by the designers and subsequently suggested modifications based on their feedback, leading to the refinement of the approach now presented. This iterative process of enhancement has been crucial for improving the device, leveraging insights from external researchers to make it more user-friendly and effective for future applications.
This brief study on drug response underscores the device's proficiency not only in concurrently recording ECGs from four distinct fish under different conditions but also in capturing multiple (four) ECG signals per fish. This multifactored capability significantly reduces the time and effort required for electrode placement and minimizes the likelihood of human error by providing a selection of signals. This choice allows for either the selection of the most accurate signal or the ability to aggregate data from multiple signals based on the study's goal. However, it's important to clarify that employing the four-electrode setup does not automatically ensure that all four signals will be clean and of high quality. The primary aim of introducing this innovative feature is to mitigate human errors and save time by enabling four concurrent recordings instead of sequential ones, which is more time-efficient and less stressful for the fish, given their limited tolerance for time out of water and under anesthesia.
<img alt="Electrophysiology setup diagram with Zebra II system, 4-electrode holder, and multielectrode array." class="xfigimg" src="/files/ftp_upload/67066/67066fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Zebra II system. (A) Isometric view of the Zebra II device (SolidWorks Model) with labeled components. <a href="https://www.jove.com/files/ftp_upload/67066/67066fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Multi-electrode ECG setup diagram and results for fish heart monitoring; channels 1-4 data shown." class="xfigimg" src="/files/ftp_upload/67066/67066fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Electrode positioning on Zebrafish. (A) The 4-point electrode holder degrees of freedom (conceptual image). (B) Electrode positioning on fish's chest. (C) ECG signal from the 4-point electrode holder (Channels 1-4). <a href="https://www.jove.com/files/ftp_upload/67066/67066fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Electrocardiogram analysis, QTc interval, ECG analysis under amiodarone dosage, heart rate charts." class="xfigimg" src="/files/ftp_upload/67066/67066fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Effects of acute amiodarone exposure (ECG signals). (A) Representative ECG data obtained from the system and its change in ECG parameters due to different Amiodarone concentrations. (B) Change in ECG due to Amiodarone exposure. (C) Bar chart describing the discrepancy of HR. (D) Bar chart describing the discrepancy QTc interval in ECG data with different Amiodarone concentrations. *p&lt;0.05 (one-way analysis of variance with Tukey test). Error bars show standard deviation. This figure has been modified from20. <a href="https://www.jove.com/files/ftp_upload/67066/67066fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current ...

zebra-ii-as-novel-system-to-record-electrophysiological-signals

Research

JoVE Journal

Bioengineering

This video shows how to perform electrocardiography (ECG or EKG) measurements, a test which measures the electrical activity of the heart, on a zebrafish.

Zebrafish Electrocardiography (ECG): A Minimally Invasive Assay to Evaluate Cardiac Function

The protocol describes the procedure to perform electrophysiology in adult zebrafish. This method can be used for measuring neural activity in vivo.

Adult Zebrafish Electrophysiology: A Method to Measure the Extracellular Electrical Activity of Neurons In Vivo

This video demonstrates the recording of electrical signals from the afferent neurons in a paralyzed zebrafish larva by positioning a micropipette near the lateral line ganglion and forming a loose seal for signal capture. The extracellular solution maintains neuronal viability during this process.

Monitoring Electrical Signals from the Afferent Neurons in an Immobilized Zebrafish Larva

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Forebrain Electrophysiological Recording in Larval Zebrafish

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

In Vivo Surface Electrocardiography for Adult Zebrafish

Discover the Zebra II system for prolonged ECG acquisition in zebrafish using multichannel electrodes. This method enables up to one hour of cardiac recording for drug screening and cardiac gene studies.

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

Explore More Videos