Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation

Our research focuses on anesthetic pharmacology, which includes an interest in anesthetic mechanisms as well as the mechanisms of anesthetic side effects. The use of larval zebrafish as a behavioral model to study anesthetic pharmacology is rapidly expanding and has many advantages, including their suitability for use in large scale screening experiments. Comparison of volatile and intravenous anesthetics remains challenging because administering volatile anesthetics to non aquatic animals is much easier than intravenous anesthetics.

Contrarily, intravenous anesthetics are more easily administered to aquatic animals than volatile anesthetics. This protocol provides a relatively inexpensive and accessible means of administering volatile anesthetics in a closed chamber to perform behavioral experiments in larval zebrafish. Other protocols of this nature often require some form of customized apparatus that can be costly or not readily accessible to those without the means to create or acquire this type of equipment.

To begin, lay out a 0.5 millimeter silicone sheet on a clean, flat surface. Inspect the sheets for any creases and avoid them when possible. Clean the sheets with 70%alcohol to remove any oil or debris.

When the sheet is completely dry, use an applicator such as a paintbrush, to apply a thin even coat of adhesive primer to the silicone sheet. Once the adhesive primer is applied, align the double-coated tape with the silicone sheet with the paper backing facing up. Gradually apply the tape to the silicone using a roller to smooth the surface and remove air bubbles.

Reapply the primer in small sections as needed, and continue the tape application as the primer dries in one to five seconds. Flip the silicone sheet over to work silicone side up to aid in visualizing and removing air bubbles. Next, cut the adhesive silicone into strips at least 10 millimeters wide.

Ensure that strips are long enough to cover at least the width of one row of the well plate. To prepare the volatile anesthetics, add a volume of the non-volatile solvent to an HPLC or scintillation vial, ensuring minimal headspace once the final dilution of volatile anesthetic is added. Use a Hamilton gas tight syringe to add the volatile anesthetic and quickly seal the vial.

Mix the vials by vortexing or sonication as needed. Temporarily store them while preparing zebrafish plates. To set up the behavioral experiment, transfer one larval zebrafish at five days post-fertilization into every other row of a glass 96 well plate.

Carefully remove the E-three solution from each well. Use a transfer pipette to fill each well in the row with the experimental solution. Using the adhesive silicone strips, press the strip downward quickly to prevent bubble formation and fish from overflowing into adjacent wells.

Then press firmly on the top of the plate to ensure each well is sealed. Repeat the sealing process until the solution is exchanged and all wells in every other row of the plate are sealed. Gently turn the plate over and inspect for air bubbles.

Place the plate into the behavior observation unit with the silicone adhesive side down, and perform the behavioral experiment. In the sealed wells, the concentration of sevoflurane exhibited minimal loss over three hours, and isoflurane showed a 4%decrease in concentration. Neither anesthetic showed significant loss during the first 30 to 60 minutes.

The median effective concentration for sevoflurane was 62 micromolar for spontaneous movement and 126 micromolar for elicited movement with the ladder being notably lower than the previously reported 240 micromolar, which may be due to improvement in creating gas tight seal with this method. A significant difference in spontaneous movement was observed between open and sealed wells, emerged at the two hour mark, likely due to the depletion of oxygen in the well, and is an important limitation of this technique.