Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation

This study describes a unique method for administering volatile anesthetics, such as isoflurane and sevoflurane, to larval zebrafish during behavioral experiments. While zebrafish offer numerous advantages as a vertebrate model organism -- including complex behaviors, genetic tractability, transparent embryos, and rapid development -- their use in studying volatile anesthetics has been limited. The administration of volatile anesthetics often requires complex or cumbersome apparatuses that may not be broadly accessible, creating barriers to the pharmacologic study of volatile anesthetics in aqueous model organisms. This method presents a straightforward technique using adhesive silicone sheets to create a gas-tight seal on glass 96-well plates. Validation was performed through the assessment of spontaneous movement, which showed no significant differences between sealed and open wells over a 90-min period. Additionally, anesthetic concentration remained stable over time, as measured by HPLC. Representative results include the experimental determination of median effective concentration (EC50) values for sevoflurane. This study provides a simple and accessible approach for pharmacologic experiments using volatile anesthetics, which can be easily adapted to study other volatile agents and experimental endpoints.