Administration of Volatile Anesthetics to Zebrafish Larvae for Behavioral Observation

Summary

This protocol presents an accessible method for creating a gas-tight environment to administer volatile anesthetics to larval zebrafish for behavioral experiments.

Abstract

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.

Introduction

Non-aquatic animal models (worms, flies, rodents, rabbits, primates, etc.) have historically been the models most commonly used to study anesthetic pharmacology. In each of these species, volatile anesthetics (administered through inhalation of gas) are far less challenging than the administration of intravenous anesthetics (administered by either intravenous or intraperitoneal routes)¹. In addition to the technical challenge of simply administering the intravenous drugs, achieving a steady state of anesthesia is far easier with volatile agents than intravenous agents².

Administration of intravenous anesthetics to larval aquatic species can be achieved by diffusion from their surrounding aqueous medium^3,4, and is thus more akin to the administration of volatile anesthetics for non-aqueous species. For many years, tadpoles (Xenopus laevis) were the model of choice for studying anesthetics due to the ease of drug administration and steady-state pharmacokinetics achieved by simple bath addition of the anesthetic to their artificial pond water^5,6. However, there are relatively few examples of the study of volatile anesthetics in this model due to the requirement of gas-tight apparatuses that are often purpose-built and can be cumbersome to use^7,8.

In recent years, the zebrafish has become a preferred model for the study of intravenous anesthetics due to the same ease of drug administration in addition to other advantages such as their well-described complex behaviors and availability of commercial units to observe these behaviors⁹. Many recent studies have employed such commercially available observation units to screen for neuroactive compounds, including anesthetics, using 96-well and 24-well plate formats to quantify the behavior of the zebrafish larvae^{4,10,11,12,13,14,15}. The larval stages are particularly well-suited for pharmacology studies due to their ability to assess many behavioral responses, their small size, high throughput compatibility, and well-characterized neural circuits^9,16,17. However, one significant limitation in these studies of anesthetics in the zebrafish model is the relative difficulty in administering volatile anesthetics. This limitation has led to scant examples of comparison between volatile and intravenous agents^4,15,18. Being able to easily study and compare both classes of anesthetics in a single animal model adds an important tool to facilitate the study of anesthetic pharmacology across all classes.

Traditionally, the study of intravenous anesthetics in an aquatic species has required specialized, often custom, equipment that can be difficult to use or may not be sufficiently gas-tight to prevent loss of the anesthetic for the duration of the experiment. The procedure described here presents an easily accessible alternative means of producing a gas-tight seal in a glass chamber that is suitable for observation of larval zebrafish behavior using a commercial observation unit (see Table of Materials).

Protocol

All zebrafish experiments were conducted in accordance with animal use protocols approved by the Institutional Animal Care and Use Committee (IACUC). Adult zebrafish were maintained at the University of Pennsylvania's aquatic facility under the oversight of the University Laboratory Animal Resources (ULAR). Tübingen long-fin wild-type zebrafish were bred in-house for all experiments and maintained under standard husbandry conditions with a 13/11-h light/dark cycle until they reached 5 days post-fertilization (dpf). Experiments were conducted using biological replicates, with clutches derived from distinct mating pairs to ensure adequate biological diversity. Details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Fabrication of adhesive silicone sheets

NOTE: Equivalents of this material (silicone sheet plus adhesive) can be commercially sourced, but often at greater expense and lesser availability.

1. Lay out the 0.5 mm silicone sheet on a clean, flat surface.
2. Inspect the sheets for any creases and avoid them when possible.
   NOTE: Silicone sheets with creases may not adequately adhere to the tape and may result in inadequate sealing of the wells and/or poor visualization of the zebrafish.
3. Clean the sheets with 70% alcohol to remove any oil or debris and allow them to dry.
4. Using an applicator, such as a paintbrush, apply a thin, even coat of adhesive primer to the silicone sheet. To avoid over-application, work in smaller sections as the adhesive promotor dries quickly (sets in 1-5 s).
   NOTE: Without this adhesion promotor, the tape will not adequately adhere to the silicone sheet.
5. Once the adhesive promotor is applied, align the double-coated tape with the silicone sheet with the paper backing facing up.
6. Gradually apply the tape to the silicone using a roller to smooth the surface and to remove air bubbles (after initial application, working silicone-side-up will aid in visualizing and removing air bubbles).
7. Allow the adhesive promoter to fully cure for 24 h on a flat surface.
8. Cut the adhesive silicone into strips at least 10 mm wide (7.5 mm well diameter plus overlap on each side).
   NOTE: When using every other row of the well plate, wider variance in strips will not negatively impact wells in adjacent rows, which may not only make application easier but also be necessary to achieve a seal around the entire circumference of the well.
   1. Ensure that strips are long enough to cover at least the width of one row of the well plate.
      NOTE: If using a well plate that is not 96-well, such as a 24-well or 48-well plate, the strip sizes can be adjusted or cut to fit the specific dimensions of the plate, ensuring proper coverage regardless of plate size.

2. Preparation of solutions

NOTE: All stock and assay solutions of volatile anesthetics were made right before the experiment to avoid loss of volatile compound concentration.

1. For each solution (stock or working solution), add a volume of non-volatile solvent (E3 embryo water, DMSO, etc.) to an HPLC or scintillation vial that when combined with the final dilution of volatile anesthetic, the total volume will sufficiently fill the vial leaving minimal headspace (see Figure 1A).
2. Add volatile anesthetic with a Hamilton gas-tight syringe and quickly seal the vial.
   NOTE: Stock solutions in vials with septum lids can be used to facilitate further dilutions via the transfer of solution with gas-tight syringes.
3. Then, mix the vials (vortex, sonication) as needed and temporarily store them while preparing zebrafish plates.

3. Setup of the behavioral experiment

NOTE: This step of the protocol may take some practice. It is recommended to work without fish or solutions containing precious material until comfortable with the technique.

1. Transfer one larval zebrafish (5 dpf) into every other row of the glass 96-well plate.
   NOTE: Leaving every other row empty allows for reduced potential for contamination of adjacent wells from spillover as the wells are sealed in step 3.4.
2. Carefully remove the E3 solution from each well. This can be done one row at a time or on the entire plate if it is able to work quickly.
   NOTE: The duration for which larvae were out of the solution was minimized to reduce potential stress. Alternative procedures may be used based on different experimental needs, including only partial removal of the solution before the addition of the drug-containing solution to minimize stress. The protocol should be adapted to suit the needs of the experiment, including equilibration time given to the animals to compensate for any potential stress, solution mixing efficiency, and accuracy of concentrations of a drug-containing solution, if not all of the E3 is removed prior to addition of the working solution.
3. Next, use a transfer pipette to fill each well in the row with the experimental solution. Take care not to introduce bubbles. Overfill each well (Figure 1B).
4. Using the adhesive silicone strips made in step 1, press the strip downward quickly (Figure 1C). This helps prevent bubble formation and keeps fish from overflowing into adjacent wells.
5. Press firmly on the top of the plate to ensure each well is sealed.
6. Repeat steps 2-5 until the solution is exchanged and the wells for every other row of the well plate are sealed (Figure 1D).
7. Gently turn the plate over and inspect for air bubbles.
   NOTE: The presence of air bubbles will obscure visualization of zebrafish movement.
8. Place the plate into the behavior observation unit (silicone adhesive side down) and perform the behavioral experiment (see the Results section for details).

Results

Comparison to traditional open-well observation
In order to assess whether adhesive sealing has any effect on spontaneous movement, a comparison was made between the controls in open versus sealed wells. As seen in Figure 2A, no statistically significant difference in movement was observed between these groups for the standard ~30 min duration used in the experiments described in this article. Recognizing that other experimental paradigms might need longer experimental e...

Discussion

This article describes a method for readily administering volatile anesthetics, like sevoflurane, to larval zebrafish, which was used to quantify volatile anesthetic potency in wild-type fish. Although this technique is conceptually simple, it can take practice to ensure an adequately sealed, bubble-free row of wells in a short time frame to minimize the loss of volatile agents. Because this method is intended for volatile compounds, it requires not only careful application of the silicone adhesive to the well plate but ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well glass microplates	Zinsser North America	3600500	"These glass microplates are made from special high purity, temperature resistant borosilicate glass and the surfaces are acid polished. Plates are rectangular in the standard SBS microplate footprint (85 x 127 mm).These glass reactor microplates are designed primarily for use with chemistry applications and are resistant to temperatures up to 530° C. They can be autoclaved. They are machined from a homogeneous glass sheet and feature uniform geometry and shape to provide consistent temperatures from well to well (within 0.2 degrees C typical)."
2.0 mL Clear, Large Opening, 9 mm Thread Vials	Chemglass Life Sciences	CV-1150-1232	"With a 40% larger opening, these vials are specifically designed to work in robotic arm auto samplers. They also incorporate the unique Step Vial design that precisely centers a limited volume insert in the neck of the vial. "
3M Double Coated Tape 9490LE	AbbVie Inc	24WG90	"3M Double Coated Tape 9490LE with 3M Laminating Adhesive 300LSE provides high bond strength to most surfaces, including many low surface energy plastics such as polypropylene and powder coated paints. The acrylic adhesive also provides excellent adhesion to surfaces contaminated lightly with oil typically used with machine parts. 3M double coated tape 9490LE offers the added feature of 3M Laminating Adhesive 300MP on one side to provide excellent bond strength to a variety of foam and fabric materials (6 in x 5 yd)"
DanioVision Observation Chamber	Noldus	DVOC-0041	"DanioVision is a complete system, designed for the high-throughput testing of zebrafish larvae in multi-well plates. It includes the Observation Chamber and renowned EthoVision XT video tracking software."
DanioVision Temperature Control Unit	Noldus	DVTCU-0011	"The DanioVision Temperature Control Unit is a flow-through system: water flows evenly underneath the well plate at the temperature of your choice. The temperature is the same all throughout the well plate."
Ethovision XT16 software	Noldus	NSE-EV-BASE	"EthoVision XT is the most widely applied video tracking software that tracks and analyzes the behavior, movement, and activity of any animal, trademarked by Noldus"
Isoflurane	Piramal Critical Care	NDC # 66794-017-10	Liquid for inhalation, a nonflammable nonexplosive inhalation anesthetic, containing 100 mL isoflurane. Stored at controlled room temperature 20º to 25º C
Scotch-Weld Instant Adhesive Primer AC79 Clear	3M	"62-3916-0860-1 (Product ID) C2103N (Lot)"	"3M Scotch-Weld Instant Adhesive Primer AC79 is designed for use on difficult-to-bond low surface energy elastomers, such as EPDM, silicone and other rubbers. Surface preparation and application are straightforward and the adhesive primer is fast drying to keep jobs moving quickly."
Silicone Sheets 0.5 mm thick	various suppliers	N/A	translucent silicone sheets were purchased from multiple suppliers
Speedball Pop-in Hard Rubber Brayer with Plastic Frame, 4 Inches"	Speedball	793728	"The Speedball Pop-In Hard Brayer has a roller that snaps out for easy cleaning. Plastic, one piece frame with detachable 4 inch wide roller. Made from pure natural gum rubber with a ground finish. The hard rubber brayer is 4 inches wide with a sturdy plastic frame and great for glue application"
SureSTART 9 mm Screw Caps, Level 2 High-throughput Applications, Type: AVCS Screw Cap Black PP, White Silicone/RED PTFE Septa 1.0 mm	Thermo Scientific	6ASC9ST1B	"Use Thermo Scientific SureSTART 9 mm Screw Caps with screw vials that have a 9 mm opening. The performance Level 2 caps are manufactured and tested to ensure low bleeding, robustness, and reproducibility of results for y routine GC-MS and LC-MS analyses. Choose from polypropylene caps with all-purpose silicone/PTFE septa of various hardness values designed to reduce autosampler needle issues. Our AVCS caps incorporate Advanced Vial Closure System technology to ensure optimal seal compression when closing a vial."
Ultane Sevoflurane	AbbVie Inc	NDC # 0074-4456-04	Volatile Liquid for Inhalation, is packaged in amber colored bottles containing 250 mL sevoflurane. Stored at controlled room temperature 15º to 30º C
Wheaton Liquid Scintillation Vials, Caps Attached to Vials, Glass, Metal Foil / Pulp, 24-400, 20 mL	DWK Life Sciences	986561	20 mL scintillation vials converted from Type I borosilicate glass tubing, PET, or HDPE. Available with cap attached.