This protocol helps simplify the measurements of electroretinograms or ERGs in larval zebrafish which provides an important functional readout of visual development and genetic disease models. The soft sponge tips of recording electrodes used in this protocol is easy to manufacture using economical and readily available materials and avoids potential damage to the larval eyes. The softer sponge-tipped electrode can facilitate repeated ERG measurements of the same eye.
Individuals new to this procedure may find it difficult to work under the dim red lighting that's used throughout the procedure. But it's important that dark adaptation is maintained. To prepare the cone-shaped sponge recording electrode, cut the end from a platinum electrode lead extension and use a scalpel to remove 10 millimeters of the outer polytetrafluoroethylene insulation coating from the new end of the extension.
Cut a 40 millimeter piece of a 0.3 millimeter diameter silver wire and entwine the silver wire with the exposed inner wire to securely attach the wire to the electrode lead. Encase the joint with insulating tape leaving an approximately 15 millimeter length of silver wire exposed. Connect another wire to the negative terminal of the battery and immerse the other end of the wire into the saline.
Immerse the exposed silver wire in normal saline and connect the other end to the positive terminal of a nine volt direct current source. After 60 seconds, place the wire on absorbent tissue to dry. In the meantime, cut a 20 by 20 millimeter square of polyvinyl acetate sponge into a cone shape and saturate the sponge in one times Ringer's buffer.
Under a light microscope with a scale bar on the eyepiece, use a scalpel blade to shape the apex of the cone to an approximately 40 micrometer diameter. Air-dry the cone-shaped sponge on absorbent tissue paper until it is solid. Before inserting the silver wire into the dried solid cone-shaped polyvinyl acetate sponge through the base of the cone, using mask tape to insulate any excess exposed metal to reduce photovoltaic artifacts.
On the day of the experiment, immerse the sponge-tipped electrode into Ringer's buffer for 15 minutes to fully saturate the sponge. At least eight hours before the recordings, place no more than 20 zebrafish larvae in a single 15 milliliter tube without a lid and wrapped in aluminum foil in a dark incubator. At the end of the incubation, pour the dark-adapted zebrafish into a petri dish under dim red illumination from a light-emitting diode.
To prepare the sponge platform, cut a piece of polyvinyl acetate sponge roughly equal in thickness to the depth of a 35 millimeter petri dish so that the sponge will fit snugly within the dish. Make a small cut vertically through one end of the sponge to accommodate the silver wire of the reference electrode and soak the sponge in goldfish Ringer's buffer until the sponge is saturated. Then place the sponge into a clean 35 millimeter petri dish and use a paper towel to absorb the extra liquid until no solution exudes from the sponge in response to a light finger press.
To position the animal for the experiment, use a three millimeter Pasteur pipette to transfer an anesthetized larva onto a three by three centimeter square of paper towel and use forceps to place the square onto the moistened sponge. Using a fine brush soaked in Ringer's buffer, adjust the position of the larva with one eye facing upward and away from any liquid on the towel under the larva. Glaze the larval body with moisturizing eye gel to keep the animal hydrated throughout the electroretinogram recording.
And place the dish on a small, water-heated platform in front of the Ganzfeld bowl light stimulus situated inside a Faraday cage. Insert the reference electrode into the cut made in the platform sponge. And connect a commercially-obtained ground electrode to the Faraday cage.
Attach the recording electrode to an electrode holder and secure the holder to the stereotaxic arm of a micromanipulator. Using a three millimeter Pasteur pipette, resaturate the sponge tip of the electrode with one drop of Ringer's solution and position the microscope in the Faraday cage over the electroretinogram platform. Use the tip of an absorbent tissue to remove any excess liquid from the electrode sponge tip and under the microscope, position the active electrode so that the sponge tip gently touches the central corneal surface of the larval zebrafish eye.
Then move the Ganzfeld bowl towards the sponge platform taking care that the larva is covered by the bowl and close the cage to reduce extraneous electromagnetic noise. The sensitivity of the larval zebrafish retina to dimmer flashes increases with age. As the a-and b-waves are not recognizable at intensities lower than minus 1.61 log candela second per meter squared at four days post fertilization while clear signals are detectable at these intensities in the older larvae.
The b-wave amplitude increases markedly between four and five post fertilization. At seven days, the signal at 2.48 log candela second per meter squared is greater compared to days five and six post fertilization. A-and b-wave implicit times become significantly faster after five days post fertilization.
With overall, these results demonstrating a maturation in zebrafish retinal function between four to seven days post-fertilization. To successfully perform this protocol it is critical that sponges are fully saturated with fluid which helps maintain the conductivity of the recording electrode and lowers the noise levels. If the a-wave is of particular interest, pharmacological treatments can be applied to block the b-wave component thus exposing the full a-wave.
