This article details a procedure for real-time monitoring of glutamate levels in brain tissue using a multielectrode array (MEA). The method involves the use of a glutamate oxidase enzyme to generate electrical signals in response to glutamate injections.
Begin with an anesthetized mouse secured on a stereotaxic frame, with the skull exposed.
Identify the skull reference points, and then mark the target coordinates.
Drill around the mark, remove the bone flap to expose the brain tissue, and clean the surface.
Attach a multielectrode array or MEA coated with glutamate oxidase enzyme and a micropipette to the stereotaxic frame.
Place the reference electrode away from the MEA while maintaining liquid contact with the brain to complete the circuit.
Fill the micropipette with a glutamate solution.
Now, Lower the MEA and micropipette into the brain tissue rich in neurons and astrocytes. Record the baseline electrical signal from the MEA.
Inject the glutamate solution to rapidly increase the glutamate levels in the extracellular space containing the MEA.
The glutamate oxidase on the MEA oxidizes the glutamate, generating an electrical signal.
As astrocytes uptake glutamate, extracellular glutamate levels decrease, reducing the signal and allowing for real-time glutamate monitoring.
In this procedure, place the animal in the stereotaxic device, and use the air bars to stabilize its head. Make sure the animal is secured and the head does not move. Then, apply eye ointment using a sterile cotton applicator. After that, shave the head with a trimmer, and remove the fur near the ears using small surgical scissors. Subsequently, apply iodine and then alcohol to the scalp three times alternatively. Following this, make an incision in the middle of the scalp, and spread the skin.
Soak up any blood with a sterile cotton tip, and apply hydrogen peroxide to facilitate the appearance of bregma and lambda. Make sure the head is properly positioned by measuring the dorsal-ventral and medial-lateral coordinates of bregma and lambda. Set the coordinates at bregma to zero, and mark the target coordinates. Next, drill around the mark. Afterward, attach the MEA to the head stage. Backfill the pipette with the first desired solution. Be sure to leave a gap in the pipette without solution, so that the solution being expelled can be examined.
Then, attach the tubing to the glass micropipette. Find bregma with the MEA attached, and set the coordinates to zero. Move the MEA to the desired coordinates and slowly lower the MEA until the tip touches the brain. Next, zero the dorsal-ventral coordinate, and then slowly lower the MEA into the brain. Place the reference electrode in a remote location from the MEA, such that it is still in liquid contact with the brain to complete the circuit. On the recording program, click on the desired calibrated electrode, and then click Perform Experiment to start recording.
After obtaining a stable baseline, set the pressure ejector to 0.6 seconds and 0.5 PSI, and press the red button on the pressure ejector to eject. Record the time and pressure as well as volume ticks moved on the injection sheet and make any notes if necessary. After that, remove the MEA with the micropipette attached. Rinse it with the eye water, and soak it in PBS overnight until all the blood is gone.
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