Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

The overall goal of this procedure is to detect and analyze ion dynamics in the extracellular space of brain.Tissue. Measurement of ion transient is accomplished by using ion selective micro electrodes, which can be prepared based on double barreled or concentric design. As a first step, the micro electrodes are calibrated in the experimental bath.

Then the electrodes are inserted into the acute mouse hippocampal slices for recording to evoke extracellular ion shifts the transmitter glutamate, or a glutamate agonist is supplied. Glutamate binds and opens ionotropic receptors at postsynaptic spines, which allows the influx of sodium into and the influx of potassium out of the cells. Ultimately, this results in a decrease in extracellular sodium as well as an increase in extracellular potassium that are detected by the ion selective micro electrodes.

This method is employed to study extracellular ion regulation and activity induced iron transients in the brain. Here we describe the preparation and calibration of two types of ion selective micro electrodes. We show how they can be employed in acute brain tissue slices to measure changes in extracellular potassium or sodium in response to the application of neurotransmitters.

The procedure will be demonstrated by Nicole Hark and Simon for my lab. To begin this procedure, prepare a bur silicate glass capillary with filament to be used for the ion sensitive barrel and another one for the later reference barrel. Next, use two component glue or a small strip of aluminum foil to fix the long and short capillary together at both ends.

Heat the double capillary in a furnace at 60 degrees Celsius for one hour to remove residual moisture. Afterward, store the electrodes in a desiccate until use center the double capillary in a vertical puller with a revolver chalk. Use a chalk to prevent elongation of the capillary.

Keep the coil gently until the glass is soft enough to allow a horizontal rotation of the revolver. Chuck by 180 degrees after cooling, remove the mechanical break. Apply a second heating protocol to pull the capillary resulting in two sharp double barreled capillaries.

The tip diameter of the double capillary should be close to one micron. Now fill the reference barrel up to its tip with distilled water to prevent colonization. In the following procedure, place a double barreled capillary with its tip up onto a custom made holder.

Then place this holder onto the wide mouth bottle containing the pre-warned HMDS and incubate for 70 minutes. After that, transfer the capillary to a metal stand. Subsequently, place it in the preheated furnace for two hours to dry both barrels after colonization, keep the capillary dry in the desiccate until use.

On the day of the experiment, prepare the ion selective electrode by filling the tip of the siliconized barrel with two to three microliters of the ion selective sensor. Then backfill the barrel with 100 millimolar sodium chloride for a sodium sensitive micro electrode or 100 millimolar potassium chloride saline For a potassium sensitive micro electrode. Be careful not to insert additional air bubbles during this procedure.

If the ization is successful and the sensor is filled properly, the sensor surface should appear as a clearly visible concave surface against the backfill. Next, fill the reference channel, then insert the chlorinated silver wires into both barrels and seal each barrel with dental wax. In this procedure, pull a two millimeter capillary in the horizontal puller to result in a short taper and a tip diameter of four microns.

Right before use, fill the siliconized capillary with 0.2 microliters of sensor. To prepare the inner capillary, pull a small diameter capillary to result in two capillaries with long tapers and sharp tips. Then fill them with 100 millimolar sodium chloride or 100 millimolar potassium chloride saline.

Place the sensor filled and the saline filled capillaries directly in line with each other onto a custom build stopper made from cover slips. Insert the smaller capillary partially into the larger capillary. Next, fix the capillaries onto another cover slip.

Using modeling clay, transfer the capillaries to the stage of a microscope and visualize them using 100 x magnification. With the help of a glass rod that is mounted on a micro manipulator, slowly advance the outer capillary over the inner capillary until the distance between their tips is roughly five microns. Fix the open end of the outer capillary onto the inner capillary using dental wax and insert silver wire.

To prepare the reference electrode, pull a capillary with filament in a vertical puller, discard the upper pipette, then open the lower chuck and lift the lower pipette by about five millimeters. Close the chuck again and lift it until the tip of the lower electrode is positioned about five millimeters above the heating coil. Subsequently heat the coil and use forceps to bend the tip by about 45 degrees to the side.

Bend it again by about minus 45 degrees to direct the tip back in parallel to the main shaft. Then fill the reference barrel with heaps buffered saline. Insert a chlorinated silver wire in it and seal the barrel with dental wax.

In this step, attach both the ion sensitive and the reference electrode to micro manipulators and lower them into an A CSF perfused experimental chamber. Under the stereo microscope, connect the chlorinated silver wires to the respective inputs of the head stage of the differential amplifier. Then determine the electrode resistances.

After that, adjust the voltage signals to zero and start recording for calibration. After obtaining a stable baseline switch to the saline containing defined concentrations of the ion to be measured. Now, transfer a 250 micron thick hippocampus slice into the experimental chamber and fix it with a grid.

Lower the calibrated ion selective micro electrode onto the slice surface. Then further lower the electrode for about 50 microns into the stratum radiator of the CA one region. To apply transmitter agonist, fill the application pipette with 0.5 millimolar glutamate.

Attach the pipette to a micro manipulator and couple it to a pressure application device. Next, lower the application pipette into the tissue and carefully place it in the proximity of the tip of the ion selective micro electrode. Start the pressure application at 40 millibars.

Shown here is the change in voltage of the reference barrels of a double barreled electrode and a concentric electrode in response to changes in the bath sodium concentration. And these are the changes in the voltage of the sodium potential of a double barreled electrode and a concentric electrode in response to changes in the bath sodium concentration. This figure shows the half logarithmic plot of the bath sodium concentration versus the voltage of the sodium potential of both electrodes.

The linear plots reveal a slope of about 48 millivolts for both electrodes. Here is the response of a double barreled and a concentric electrode to a fast change in the bath. Sodium concentration from 152 millimolar to 70 millimolar.

Note that the response time of the concentric electrode is significantly faster. This figure shows the extracellular sodium transient as detected by double barreled and concentric electrodes, transient changes in the voltage of reference barrels and the extracellular sodium concentration were induced by bath perfusion with 10 millimolar aspartate for 120 seconds as indicated by the bar. And in this figure transient changes in the voltage of reference barrels and extracellular sodium concentration were induced by local pressure application with 10 millimolar glutamate for 0.2 seconds.

Note that the response time of the conal electrode is significantly faster under this condition. While attempting this procedure, it's important to keep in mind that proper silent is the key step in the successful production of iron elective micro electrodes. After watching this video, you should have a good understanding of how to prepare and use these electrodes for the detection of extracellular iron, transient in brain tissue.