The overall goal of this procedure is to obtain in vivo recordings of neural activity in the brain of intact adult zebrafish. This is accomplished by first obtaining an immobilizing, an adult zebra fish through the bath application of Trica Methyls sulfonate. The second step is to intubate the immobilized zebra fish.
Next, a craniotomy is performed under trica anesthesia to provide access to the brain. The final step is to position a primary electrode within the craniotomy window, which allows extracellular brain activity to be recorded. Ultimately in vivo.
Electrophysiology in the adult zebrafish can be used to show how different pharmacological compounds or genetic mutations can lead to alterations in the native neurological activity of the zebrafish. This method can help answer key questions in both developmental biology and neurobiology about how changes in brain structure can affect changes in neural activity, both in normal development and in the case of genetic mutation. Generally, individuals new to this technique will struggle because extra care must be taken to remove only a small portion of the bony plate covering the head without damaging the brain tissue.
The intubation cannula facilitates the introduction of fluid to the fish. This setup provides both flexibility and firmness to best position the cannula within the animal's mouth. For this setup, remove a 1.5 centimeter section from the wide end of a P 200 pipette tip with a pair of scissors or a razor blade.
Next, insert a six centimeter by one millimeter piece of tubing into a reducing valve. Then insert the modified pipette tip into a short portion of one eighth of an inch diameter tubing. The cannula can then be inserted into the pipette tip using the tubing to hold the pipette tip in position with the lure lock.
Before the experiment flushed a profusion setup with habitat water, ensuring that all the air bubbles are removed. Next, add 30 milliliters of 630 micromolar trica solution to the craniotomy perfusion setup, and allow two milliliters of it to run through the tubing to ensure the delivery of trica to the animal upon profusion initiation. For the main profusion setup, add 50 milliliters of habitat water to the first tube.
Then add any desired experimental compounds to each of the remaining tubes. After that, place a cannula setup into the small hole of the intubation base twist a one inch wide strip of Kim wipe into a tight spiral and insert it into the drainage tube such that the Kim wipe extends on both ends. This tissue will serve as a wick to remove the perfused liquid and to prevent fluid buildup within the intubation base that could interfere with electrophysiological recording.
Next, insert one end of the tubing into the large hole of the Petri dish, allowing the lower end to extend into an inert dish that is large enough to collect all profusion waste. This dish will serve as the outflow reservoir for the setup. Then position the tissue so that one end will lie near the animal's abdominal region, and the lower extension can drip into the dish.
Place this completed intubation base near the dissection microscope. Subsequently connect the lu lock of the cannula to the trican profusion tubing. In this procedure, add 630 micromolar trica solution to a 15 by 60 millimeter Petri dish up to three quarters.
Next, weigh the fields dish. This will be used to immobilize the animal so that further anesthesia can be injected into peritoneal. Now immerse the animal into the dish containing trica and weigh the dish again.
Then calculate the weight of the fish by subtracting the weight of the filled dish from the total weight. Allow the animal to remain in the trica solution until it is calm and most movement has ceased. While the fish is immersed in the trica solution, use a NFI syringe containing a 34 gauge needle to withdraw enough pantonium bromide such that there is one microgram per gram of fish weight.
Once the fish is calmed, use a pair of broad forceps to transfer the fish by the tail to a pre-moistened sponge and position it laterally. Next place a sponge and the fish under the dissecting microscope. Hold the dorsal side of the fish with a popsicle stick.
Then administer the pan keon bromide interperitoneal. Now use fine forceps. Grab the fish by the lower jaw and quickly transfer the animal to the intubation base.
Position the animal dorsal side up on the wax form such that the one millimeter cannula can be inserted into the mouth. Next, use the fine forceps to maneuver the fish and open the mouth around the cannula. In our setup.
A stop was engineered into the base, allowing the cannula to be inserted three millimeters into the mouth of the fish. Once it is in position, turn on the gravity fed perfusion tube containing the trica. Then cut a three square centimeter section of Kim wipe tissue and wed it with habitat water.
Place the tissue over the animal to prevent desiccation. Under the dissection microscope, the fish is prepared and waits to have the craniotomy carried out. This area looks like a dark bony plate that sits behind the eye.
Next, insert one blade of the van of scissors at the edge of the plate and push it with enough force to penetrate the bone without piercing the brain. Then close the scissors to snip the bone. After that, remove the piece of bone for electrophysiology recording.
Shut off the intubation. Stop cock and quickly disconnect the lure lock connecting the intubation base to the trica drip. Move the intact intubation setup to the electrophysiology microscope and connect it to the perfusion system.
Turn on the habitat, water and perfuse at a rate of one milliliter per minute for about one hour. In order to wash out the trica using a micro manipulator, position the secondary electrode so that the electrode tip can be inserted into the animal's nostril or into the dip under the upper jaw. Subsequently, insert the primary electrode needle into the craniotomy opening.
Then insert the needle into the tissue such that the tip is positioned fairly superficial within the optic tum. Deeper positions of the electrode may result in a small electrical signal for data collection gap free mode at 5, 000 hertz sampling rate, together with a low pass filter of 0.1 kilohertz and a high pass filter of one hertz are used. Begin the recording of the electrophysiological activity when the habitat water has perfused for a minimum of 45 minutes.
Record a baseline of native activity for at least 15 minutes prior to the addition of experimental drugs. The native neurological activity of the adult zebrafish was monitored in this experiment, this figure shows the spontaneous baseline activity of the optic tum with a small amplitude around two to 10 millivolts throughout the duration of the recording. Following the introduction of 15 millimolar PTZ to the system, spontaneous epileptic form like discharges began to develop within five minutes.
Continuous exposure to PTZ led to a consistent pattern of up to 15 millivolt, large amplitude discharges, followed by smaller, more frequent bursts of activity. While carrying out this procedure, it is important to remember to be patient and to take your time while handling the animal and carrying out the craniotomy. After watching this video, you should have a good idea of how to obtain in vivo recordings of neural activity in the brain of an intact adult zebra fish.
This is accomplished by using a profusion system and performing a craniotomy on the immobilized animal.
