Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

The overall goal of this procedure is to measure tonic and phasic extracellular glutamate changes in vivo using enzyme linked MicroElectrode Arrays. This method can help answer key questions in the neuroscience field, such as whether extracellular neurotransmitter levels and the fast dynamics of neurotransmitter release and clearance are altered in disease states. The main advantage of this technique is that the MicroElectrode Arrays can measure specific neurotransmitter changes within discrete brain regions, making it a spatially and temporally precise approach while being minimally invasive.

Demonstrating the procedure will be Holly Hunsberger and Ryan Heslin from my laboratory. To begin this procedure, coat a pair of recording sites on an MEA with the desired enzyme and a different pair of recording sites with the inactive protein matrix. Then, draw up the glutamate oxidase or protein matrix using a 10 microliter Hamilton syringe.

Gently press the plunger to dispense a small bead of solution at the syringe tip. To target the MEA recording sites, under a dissecting microscope, apply the solution to the appropriate recording sites by briefly contacting the recording sites with the solution droplet, which represents one layer. Set a timer for one minute between coating applications to the recording sites.

Now, place the reference electrode through the opening in the plastic arm. Lower the reference electrode into a beaker containing PBS and ensure that it does not contact the bottom of the beaker. Next, connect the reference electrode to the head stage and connect the MEA to the mPD plated to the head stage.

Lower the MEA into the beaker of solution such that only the MEA tip is submerged in solution. Do not submerge the MEA tip in liquid beyond the black bubble. Doing so may damage the MEA.

Then, select the electroplating icon on the desktop. In the electroplating tool menu, verify the correct settings are entered. Afterward, select exit software to finish electroplating.

Rinse the mPD plated electrode tips with DI water and store for 24 hours before calibrating. In this procedure, turn on a heating pad at 37 degrees Celsius and connect the head stage to the recording control system. Then, insert the MEA tip into 40 milliliters of stirred 0.5 molar PBS.

Next, connect the reference electrode to the head stage. Flick the reference electrode tip to ensure there are no air bubbles. After that, open the recording program, make sure the settings are correct, choose the MEA number, and click Calibration, and press start.

Allow five to 10 minutes for equilibration and begin calibration once the baseline has stabilized. Next, select Baseline and add 500 microliters of ascorbic acid. Then, select Interferent once the current has reached a new steady plateau.

If the MEA is properly electroplated, no change should be observed. Next, add 40 microliters of 20 millimolar L-glutamate. Once the current has reached a new steady plateau, mark first addition as analyte.

Repeat three times for a total of three glutamate additions. Then, add 40 microliters of dopamine. And select Test Substance.

Subsequently, add 40 microliters of peroxide. Select Test Substance and click the stop button once the calibration is finished. In this step, place the micropipette centrally among all four platinum recording sites and mount it 50 to 100 micrometers above the MEA using sticky wax and modeling clay.

Using a DC adapter, clamp the red positive wire to the prepared silver wire on the gold pin side, and clamp the black negative wire to the platinum bath cathode. Next, plug in the DC adapter and look for the correct plating process. Bubbles should appear at the bath electrode, and the reference electrode should turn a silver gray color.

In this procedure, place the animal in the stereotaxic device and use the ear 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 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.

Back fill 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's 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 DI water, and soak it in PBS overnight until all the blood is gone. In the CA3 and CA1 regions of the hippocampus, tonic glutamate levels were significantly increased in the vehicle treated TauP301L mice, an effect attenuated by riluzole treatment.

Here, the baseline matched representative recordings of potassium chloride evoked glutamate release in the CA3 showed riluzole treatment attenuated the significant increase in the amplitude of glutamate release observed in Vehicle-TauP301L mice. Local application of potassium chloride produced a robust increase in extracellular glutamate that rapidly returned to tonic levels. The significantly increased potassium chloride evoked glutamate release in the DG, CA3, and CA1 observed in the Vehicle treated TauP301L mice after local application of potassium chloride was attenuated with riluzole treatment.

This cresyl violet stained section of hippocampus confirms the location of MEA tracks in CA3 and CA1. While attempting this procedure, it's important to remember the troubleshooting steps listed in the manuscript. For instance, to reduce noisy signals, remember to ground the system and follow the steps in a timely manner.