Measuring Brain Glutamate Levels Under Anesthesia in Neonatal Piglets Using a Microelectrode Array

Secure an anesthetized neonatal piglet to a stereotaxic frame.

Make a midline incision to reveal the skull.

Remove a portion of the skull, then remove the dura to access the brain.

Fix a microelectrode array, or MEA to the stereotaxic frame.

The MEA consists of a rigid shaft and recording sites, including glutamate-sensitive sites coated with glutamate oxidase and sentinel sites coated with an inactive protein matrix.

Position the MEA vertically over the surgical site and place a pseudo-reference electrode under the scalp for a stable baseline voltage reference.

Insert the MEA into the brain region of interest and initiate measurement.

Glutamate oxidase on the recording sites converts extracellular brain glutamate to hydrogen peroxide, which undergoes electrochemical oxidation at the electrode surface, generating current.

The sentinel sites measure background noise.

Normalize the glutamate-sensitive site signals to the sentinel site signals to measure the brain glutamate levels under anesthesia.

To begin, create a 4- to 6-centimeter midline incision along the skull using caution to avoid scoring the skull with the scalpel. Once the incision is made, use gentle retraction and blunt dissection to elevate the scalp from the skull.

Next, gently scrub the skull with a gauze pad to remove any connective tissue and expose the suture lines. Then, determine the intended location for the craniotomy. If the area of interest remains obscured, further reflect the scalp.

Now, use a surgical drill to create a craniotomy window that is about 0.25 square centimeters overlying the structure of interest. Be careful not to injure the dura or the underlying brain. As needed, use fine surgical tools to excise the dura overlying the brain tissue. Use extreme care to avoid damaging the brain.

This experiment utilizes a previously described enzyme-based microelectrode array precoated with glutamate oxidase and electroplated with MPD. The microelectrode arrays have a 40-millimeter rigid shaft customized for use with piglets. Secure the metal arm to the micromanipulator, and then position the microelectrode array as vertically as possible over bregma.

Then, carefully lower the array as low as possible without touching the surface of the skull, noting the coordinates of bregma. Now, use a piglet brain atlas to determine the exact stereotaxic coordinates of the structure of interest. Then, reposition the microelectrode accordingly.

Next, place the pseudo-reference electrode under the scalp, ensuring contact with the animal. Now, slowly lower the microelectrode array into the brain to nearly the appropriate depth. For the final 2 millimeters of travel, use a hydraulic microdrive to gently lower the array into the structure of interest with minimal tissue trauma.

After the microelectrode array is positioned, wait 30 minutes to allow the electrodes to reach baseline. Then, take measurements for about three hours. If the piglet is to survive the experiment, close the incision after collecting data.