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Development of a novel approach using the microelectrode array to measure neural activity and network function in larval zebrafish
Citation:
Keever, J., A. Carpenter, J. Hedge, S. Padilla, T. Shafer, AND M. Martin. Development of a novel approach using the microelectrode array to measure neural activity and network function in larval zebrafish. Society of Toxicology 62nd Annual Meeting and ToxExpo 2023, Nashville, TN, March 19 - 23, 2023. https://doi.org/10.23645/epacomptox.22652287
Impact/Purpose:
Poster presented to the Society of Toxicology 62nd Annual Meeting and ToxExpo March 2023. Assessment of Developmental Neurotoxicity (DNT) using animal-based Guideline studies is expensive, time consuming, and does not always yield actionable data. Further, DNT assessment is not required under TSCA and the Guideline DNT study is a triggered study under FIFRA. Consequently, few of the tens of thousands of chemicals to which humans may be exposed have been evaluated for DNT hazard. To address this issue, New Approach Methodologies (NAMs) are being developed that can help provide data for risk decisions under FIFRA and TSCA. As these methods become more widely utilized, they can also provide information for other types of environmental decision-making as well. We describe here a novel approach with zebrafish that will link in vitro assessments with in vivo behaviors.
Description:
Traditional in vivo developmental neurotoxicity (DNT) testing is resource intensive. Given the number of chemicals with unknown DNT hazard potential, New Approach Methodologies (NAMs) are being developed with an emphasis on high-throughput in vitro and alternative animal models. Zebrafish have been proposed as an alternative-animal model for DNT chemical hazard assessment. The larval zebrafish photomotor response assay has been utilized as a DNT NAM to screen for potential chemical hazard. In addition, microelectrode array (MEA) technology has been used as an in vitro DNT NAM to measure spontaneous neural activity and network formation. Here, we have developed an in vivo zebrafish MEA assay which attempts to connect the Zebrafish photomotor response and MEA technology to support the use of DNT NAMs. Zebrafish were collected at ~4h post-fertilization and incubated until 6 days post-fertilization (DPF). A protocol was then developed and optimized for embedding 6 DPF Zebrafish larvae into a 6-well MEA plate using low-melt agarose. Neural activity was recorded using the Axion BioSystems’ Maestro MEA system. Results indicate that zebrafish neural activity was consistent over a 10-minute recording period and a power analysis indicates that a sample size of 15 zebrafish is sufficient. To provide proof-of-concept, zebrafish were treated with picrotoxin and tetrodotoxin, as these chemicals have known effects on neural activity. Acute concentration-response experiments (0.1µM to 100µM) were performed and indicate significant changes in Zebrafish MEA activity at the 100µM concentration. At 100 µM, both number of spikes (action potentials) and mean firing rate (Hz) were significantly different to control (non-treated) zebrafish. To incorporate alternating light-dark photoperiods (simulating the zebrafish photomotor response assay), an LED box with adjustable lighting was constructed to precisely fit above the MEA plate during recording. Multiple light intensities (300, 600, 1200, and 2400 lux) were tested to establish the light intensity that induced the most robust zebrafish response. Electrophysiological responses to different light intensities were not found to be significantly different from each other. However, there were trends to suggest that the lowest light intensity (300 Lux) induced the most consistent and robust response. Next, the duration of the alternating photoperiod intervals were evaluated. Here, four photoperiod lengths (2, 3, 5, and 10 min) were tested. The different photoperiod lengths were found to have a significant effect on Zebrafish MEA activity when analyzing the 10-second period following a photoperiod change. Based on these and previous findings, zebrafish light-dark MEA experiments were optimized to use 335 lux and 2-minute photoperiods. Assessing positive and negative DNT control chemicals with the larval zebrafish light-dark MEA assay will allow for the direct comparison of MEA data with the larval photomotor response assay. Ultimately, these approaches will help to bridge the gap among DNT NAMs and will support their use for chemical hazard identification and risk assessment. This abstract does not necessarily reflect U.S. EPA policy.
URLs/Downloads:
DOI: Development of a novel approach using the microelectrode array to measure neural activity and network function in larval zebrafish
ZEBRAFISH PIP POSTER_FINAL_CLEARANCE.PDF (PDF, NA pp, 1259.144 KB, about PDF)