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

This study was carried out in strict accordance with the institutional guidelines of the Heinrich Heine University Düsseldorf, Germany, as well as the European Community Council Directive (86/609/EEC). All experiments were communicated to and approved by the Animal Welfare Office at the Animal Care and Use Facility of the Heinrich Heine University Düsseldorf, Germany (institutional act number: O52/05). In accordance with the German Animal Welfare Act (Tierschutzgesetz, Articles 4 and 7), no formal additional approval for the post-mortem removal of brain tissue was necessary.

1. Preparation of Double-barreled Ion-selective Microelectrodes

1. Prepare two borosilicate glass capillaries with filament: one with a length of 7.5 cm and an outer diameter of 1.5 mm to be used for the ion-sensitive barrel, and one with a length of 6.5 cm and an outer diameter of 1.0 mm for the later reference barrel.
2. Clean the capillaries in acetone for 12 hr and then rinse several times with abs. ethanol and let them dry. Electrodes can now be stored in a desiccator.
3. Use two-component glue or a small stripe of aluminum foil to fix a long and a short capillary together at both ends. Heat the double-capillary in a furnace at 60 °C for 1 hr. This accelerates the curing process. Store the electrodes now in a desiccator.
4. Center the double-capillary into a vertical puller with a revolvable chuck. Heat the coil gently until the glass is soft enough to allow a horizontal rotation of the revolvable chuck by 180°. During this step, prevent elongation of the capillary with a chock under the chuck.
5. After cooling, remove the mechanical break and apply a second heating protocol to pull out the capillary, resulting in two sharp, double-barreled capillaries (Figure 1). The tip diameter of one double-capillary should be close to 1 µm.

Figure 1. Architecture of ion-selective microelectrodes. (A) Photograph of a double-barreled microelectrode. For illustration purposes and better visibility, the liquid inside the reference barrel was tinted. (B) Photograph of a concentric microelectrode and the corresponding reference electrode. The tip of concentric microelectrode is shown enlarged at its left. In (A) and (B), the space occupied by the ion sensor in the tips of the pipettes is post-colored. At the bottom, the tip arrangement of both electrode types is shown schematically. Please click here to view a larger version of this figure.

6. If a vertical puller with revolvable chuck is not available, prepare a double-barrelled ion-selective microelectrode in a horizontal puller using theta glass.
   1. To this end, pull theta glass (outer diameter of 1.5 mm, length 7.5 cm) out to result in two electrodes with two barrels each. Mark one of them to serve as the future reference barrel. Silanized the other similar to the procedure described below to serve as the future ion-selective barrel.
      NOTE: While the preparation of double-barreled theta-glass electrodes is far easier than the preparation of twisted double-barreled electrodes, they are more prone to cross-action between both barrels as the thin separating glass wall between them tends to be porous in their utmost tip.
      NOTE: Because the sensor cocktail (see below) is hydrophobic, the inner surface of the later ion-sensitive barrel must be rendered hydrophobic as well by a process called silanization. For this, hexamethyldisilazane (HMDS) is used (Figure 2) as described in the next step.

Figure 2. Silanization of pipettes. (A) Hexamethyldisilazane (HMDS) reacts with the Si-OH groups of the inner glass surface and renders it hydrophobic. (B) The left photograph shows the entire silanization unit. A wide-mouth bottle containing HMDS is placed on top of a heating plate set at 40 °C. On top of the bottle, a holder (PH) carrying the capillaries (Cap) is mounted. The right photograph shows an enlargement of the pipette holder (PH) carrying the capillaries (Cap). (C) Schematic side view of the custom-made pipette holders (PH) for double-barrelled (left) or concentric (right) capillaries (Cap) as mounted on a wide-mouth bottle. Please click here to view a larger version of this figure.

7. Just before pulling electrodes, fill approx. 20 ml HMDS into a wide-mouth bottle. Attention: HMDS is for use in a fume hood only, vapors are hazardous to health! Close the bottle and put it onto a heating plate, preset to 40 °C. Preheat a furnace, placed under the hood as well, up to 200 °C.
8. Fill the reference barrel up to its tip with distilled water to prevent its silanization during the following procedure.
9. Put double-barreled capillary with its tip up onto a special, custom-made holder (Figure 2B, 2C). The blunt open end must reach freely through the bottom of the holder (Figure 2C, left). Afterwards, place this holder onto the wide mouth bottle containing the pre-warmed HMDS for 70 min.
   NOTE: Make sure that HMDS vapor can flow through capillaries freely. During one silanization process, the 20 ml of HMDS do not evaporate completely, and the bottle can thus be used multiple times.
10. Afterwards, transfer the capillaries to a metal stand and into the preheated furnace for two hrs. This will dry both barrels.
11. After silanization, keep the capillaries dry until use. While the former procedure requires about 3.5 hrs in total, electrodes can now be stored in a desiccator for several weeks.
12. On the day of their experimental use, prepare ion-selective electrodes by filling the tip of the silanized barrel with 1-2 µl of the ion-selective sensor (Figure 1A). For potassium-sensitive microelectrodes, use the carrier valinomycin; and for sodium-sensitive microelectrodes, use ETH 157 as carrier.
13. Observe that the ion-selective sensor is quite viscous making it difficult for the tip to fill properly.
14. Fill the reference barrel with HEPES-buffered saline (see below).
    NOTE: While other, more simple iso-osmotic saline could be used as well, we prefer to use a saline the composition of which is essentially similar to the perfusion saline (ACSF, see 3.6.), because it may leak out and penetrate into the tissue during experiments. Therefore, it is also essential that this saline does not contain a higher concentration of the ion to be determined than the ACSF used.
15. Backfill the sensor with saline that contains a high concentration of the ion to be detected. Thus, backfill the sensor with 100 mM NaCl saline (for sodium-sensitive microelectrodes) or 100 mM KCl (for potassium-sensitive microelectrodes). Be sure not to insert additional air bubbles during this procedure.
16. If the silanization was successful and the sensor is filled properly, observe the sensor surface form a clearly visible concave surface against the backfill (Figure 1A). Otherwise, the sensor may have retracted from the capillary wall and the tip will not be filled. Such electrodes will not be functional.
17. Insert chlorinated silver wires into both barrels (be careful not to touch the sensor) and seal each barrel with dental wax (Figure 1A).

2. Preparation of Concentric Ion-selective Microelectrodes

1. For the outer barrel, use thin-walled glass capillaries with filament (outer diameter 2.0 mm) and a length of 7.5 cm. For the inner barrel, take thin-walled capillaries with filament, an outer diameter of 1.2 mm and a length of 10 cm.
2. Clean the capillaries in acetone for 12 hrs. and then rinse several times with abs. ethanol and let them dry. Electrodes can now be stored in a desiccator.
3. Fill approx. 20 ml HMDS into a wide-mouth bottle. CAUTION! HMDS is for use in a fume hood only, vapors are hazardous to health. Close the bottle and put it onto a heating plate, preset to 40 °C. Preheat a furnace, placed under the hood as well, up to 200 °C.
4. Insert the 2.0 mm capillary into a horizontal puller and pull it out to result in capillaries with short tapers and a tip diameter of ~4 µm (Figure 1B).
5. Put pulled-out 2.0 mm capillary with its tip up onto a custom-made, special holder (Figure 2B, Figure 2C) so that its blunt end opens to the cavity of the bottle (Figure 2C, right). Afterwards place this holder onto the wide mouth bottle containing the pre-warmed HMDS for 70 min. Make sure that HMDS vapor can flow through all capillaries.
6. Afterwards, transfer the capillaries to a metal stand and into the preheated furnace for two hrs.
7. After silanization, keep the capillaries dry until use. While the former procedure requires about 3.5 hrs in total, now store the electrodes in a desiccator for several weeks.
8. Just before use, fill a small amount of sensor (0.2 µl) into the silanized capillary (Figure 1B).
9. To prepare the inner capillary, insert a small diameter capillary into the puller and pull out to result in two capillaries with long tapers and sharp tips (Figure 1B). Fill with 100 mM NaCl (sodium-sensitive microelectrodes) or 100 mM KCl (potassium-sensitive microelectrodes) saline.
10. Place the sensor-filled and the saline-filled capillary directly in line with each other onto a custom build stopper made from coverslips. Insert the smaller capillary a short way into the larger capillary.
11. Fix the capillaries onto another coverslip using modeling clay, transfer to the stage of a microscope and visualize using 100x magnification. With help of a glass rod that is mounted on a micromanipulator and by turning the fine drive of the stage of the microscope, slowly advance the outer capillary over the inner capillary until the distance between their tips is roughly 5 µm (Figure 1B).
12. Fix the open end of the outer capillary onto the inner capillary using dental wax. Alternatively, apply a small drop of cyanoacrylate (Crazy Glue) instead of wax.
    NOTE: Addition of a small droplet of water will polymerize the glue and fix the electrodes in place. Insert a chlorinated silver wire into the inner barrel and seal it with dental wax (Figure 1B).
13. To prepare the reference electrode, take a 7.5 cm long capillary with filament and an outer diameter of 1.5 mm and pull out with a vertical puller. Ensure that the tips are relatively long but not too sharp (tip diameter ~1 µm).
14. Discard the upper pipette, open the lower chuck and lift the lower pipette by about 5 mm. Close the chuck again and lift it until the tip of the lower electrode is positioned about 5 mm above the heating coil. Heat the coil and use forceps to bend the tip by about 45° to the side. Lower pipette about 1-2 mm. Bend again by about -45° to direct the tip back in parallel to the main shaft (Figure 1B). This procedure is necessary to enable the close positioning of the tip of the reference electrode to the concentric electrode.
15. Fill the reference barrel with HEPES-buffered saline (see below), insert a chlorinated silver wire and seal the barrel with dental wax (Figure 1B).

3. Salines

1. Prepare saline for backfill of potassium-sensitive electrodes (see 1.15.) composed of 100 mM KCl. Saline for backfill of sodium- sensitive electrodes is composed of 100 mM NaCl.
2. Prepare saline for backfill of reference barrels (HEPES-buffered saline, see 1.16.) is composed of (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 2 MgSO₄, 1.25 NaH₂PO₄, and 25 HEPES , titrated with NaOH to result in a pH of 7.4.
3. Prepare saline for calibration of potassium-sensitive electrodes are composed of 25 mM HEPES and a total of 150 mM NaCl and KCl, pH adjusted to 7.4 with NMDG-OH (N-methyl-D-glucamine). Here, calibration was performed with salines containing 1, 2, 4 or 10 mM KCl; ACSF contained 2.5 mM KCl (Figure 5).
4. Prepare saline for calibration of sodium-sensitive electrodes as follows; (in mM) 25 HEPES, 3 KCl, a total of 160 NaCl and NMDG-Cl, pH adjusted to 7.4 with NMDG-OH. Perform calibration with saline containing (in mM) 70, 100, 130 or 160 NaCl; ACSF contained 152 NaCl (Figure 6).
5. For determination of cross-reaction of the sensor with other ions, e.g., pH, prepare additional calibration salines with fixed ion concentrations, but varying pH.
   NOTE: While this procedure is not demonstrated in the present study, please should refer to earlier work addressing this issue (e.g. ^11,12).
6. Prepare acute brain tissue slices in saline composed of (in mM): 125 NaCl, 2.5 KCl, 0.5 CaCl₂, 6 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 glucose, bubbled with 95% O₂ and 5% CO₂, resulting in a pH of 7.4.
7. Perform experiments in brain slices in artificial cerebrospinal fluid (ACSF) composed of (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 glucose, bubbled with 95% O₂ and 5% CO2, resulting in a pH of 7.4.
8. For stimulation of neurons and glial cells, prepare solutions containing 0.2, 0.3, 0.4 or 0.5 mM glutamate or 10 mM L-aspartate dissolved in ACSF. For pressure application, dissolve 10 mM glutamate in HEPES-buffered saline.
9. Prepare application pipette for pressure application. Insert thin-walled glass capillaries with filament (outer diameter 2.0 mm) and a length of 7.5 cm into horizontal puller and pull out to result in a tip diameter of ~ 1 µM.
10. For induction of epileptiform activity, prepare magnesium-free ACSF containing 10 µM bicuculline methiodide.
11. To inhibit the generation of action potentials, prepare a 10 mM stock solution of tetrodotoxin (TTX) in distilled water. During experiments, TTX is directly added to the ACSF to result in a final concentration of 0.5 µM.
12. To prevent activation of AMPA-receptors, prepare a 50 mM stock solution of cyano-nitroquinoxaline-dione (CNQX) in dimethyl sulfoxide (DMSO). During experiments, this is directly added to the ACSF to result in a final concentration of 100 µM.
13. To block activation of NMDA-receptors, prepare a 50 mM stock solution of amino-phosphonopentanoate (APV) in 70 mM NaHCO₃. During experiments, this is directly added to the ACSF to result in a final concentration of 100 µM.

4. Calibration of Ion-selective Microelectrodes

1. Calibrate ion-selective microelectrode directly before and after each experiment.
2. Attach electrodes to micromanipulators and insert them into an ACSF-perfused experimental chamber, placed under a stereo microscope (Figure 3, 4). Place the reference electrode into the pre-chamber (Figure 4A, B). Switch on bath perfusion, ensure that the flow is about 2 ml/min.

Figure 3. The experimental work space. The recording rig consists of a vibration-damped table carrying the x/y-translational stage with the experimental bath, the micromanipulators, and a stereomicroscope with high quality optics. The stereo microscope is also equipped with a CCD camera for documentation purposes. In addition, a pressure application device for focal drug application is used. The recording electrodes are coupled via the head stage to a differential amplifier. The digitized data (A/D converter) is recorded with a computer. The bath perfusion is realized by a peristaltic micro pump (not shown). Please click here to view a larger version of this figure.

3. Switch on the A/D converter, the differential amplifier, and the computer, and start the recording software (Figure 3, 4). Because resistance of the ion-sensitive barrels is very high (10-20 GΩ; see below), use a special electrometer amplifier with high input impedance (R_videodan= 10 TΩ) and a small bias current (I_bias= 50 fA – 1 pA).

Figure 4. Electronic and spatial design of the experimental setup. (A) Schematic view of the recording arrangement. The tips of a double-barreled microelectrode (blue) and a concentric microelectrode (red) are arranged in an experimental bath filled with saline. The bath reference electrode is indicated schematically. The potential detected by the ion-selective barrels (V₁) is composed of the electrical field potential (V_ref) and the ion potential (V_ion), whereas the reference barrels (V₂) only detect V_ref. To isolate V_ion, V_ref is subtracted from V₁ by means of a differential amplifier (D.A.). (B) Topography of the experimental stage with a concentric microelectrode in place. The center of the experimental stage consists of the experimental bath containing the slice preparation. To ground the bath, the reference bath electrode is submerged in the pre-chamber which also hosts the perfusion inlet. The stage itself is grounded by a separate ground connector. The concentric microelectrode and its reference electrode are carried by separate pipette holders driven by micromanipulators. Microelectrode and reference electrode are electronically coupled to the head stage of the amplifier. Please click here to view a larger version of this figure.

4. Connect chlorinated silver wires to the respective inputs of the head stage of the differential amplifier (Figure 4). Determine electrode resistances. Ensure that the resistance of the reference barrel is between 30-100 MΩ and the resistance of the ion-sensitive barrel is between 10-20 GΩ.
5. Adjust voltage signals to zero and start recording.
6. Note that the potential detected by the ion-selective barrels (V₁) is composed of the electrical field potential (V_ref) and the ion potential (V_ion), whereas the reference barrels (V₂) only detect V_ref. To isolate V_ion, V_ref is subtracted from V₁ by means of a differential amplifier (D.A.) (Figure 4A).
7. After obtaining a stable baseline, switch to calibration salines containing defined concentrations of the ion to be measured.
   NOTE: Figure 5A shows the calibration of a double-barreled potassium-sensitive microelectrode. In Figure 6A, the calibration of both a double-barreled as well as a concentric Na⁺-sensitive microelectrode is shown. While we do not describe the procedure here, note that possible interferences with other ions must be tested before using a specific ionophore (see also 3.4.).

Figure 5. Calibration of double-barreled potassium-selective microelectrodes. (A) Change in the voltage of the reference barrel (V_ref) and of the K⁺-potential (V_K+) in response to changes in the bath K⁺ concentration ([K⁺]_b) as indicated. (B) Half-logarithmic plot of [K⁺]_b versus V_K+. A linear plot of the data reveals a slope of about 56 mV. For illustration purposes, traces were smoothed with a Sawitzky-Golay filter (width 20). Please click here to view a larger version of this figure.

8. Determine the voltage response of the ion-selective barrel in response to a known change in ion concentration. Plot data in a single logarithmic plot (Figure 5B, 6B) and determine the slope. An ideal sensor follows a relationship described by the Nernst equation (see e. g. ¹³):

V: measured electrode potential;
V₀: standard electrode potential, e. g. for a AgCl-wire at a temperature of 298.15 K and a partial pressure of 101.325 kPa (absolute)
R: gas constant (8.314 joules/degree Kelvin/mole)
T: absolute temperature (in Kelvin)
z: charge on the ion (+ 1 for potassium and sodium)
F: Faraday constant (96.500 coulombs)
c’: extracellular ion concentration
c’’: intracellular ion concentration

For practical purposes, and the natural logarithm are converted to the Nernst slope s which is then valid for a given ion and temperature:

NOTE: For potassium and sodium electrodes, an ideal voltage response of the sensor thus exhibits a linear slope of about -58 mV at RT. Some deviation of this can be accepted (Figure 5B, 6B). It is essential, however, that response characteristics of a given electrode do not change significantly (by more than 10%, see below) before and after an experiment.

Figure 6. Calibration of sodium-selective microelectrodes and comparison of double-barreled with concentric electrodes. (A) Top traces: Change in the voltage of the reference barrels (V_ref) of a double-barreled electrode (black dotted trace) and a concentric electrode (gray trace) in response to changes in the bath Na⁺ concentration ([Na⁺]_b) as indicated. Note that the voltage responses of both electrodes are virtually identical. Bottom traces: Changes in the voltage of the Na⁺-potential (V_Na+) of a double-barreled electrode (black trace) and a concentric electrode (grey trace) in response to changes in [Na⁺]_b. (B) Half-logarithmic plots of the [Na⁺]_b versus the V_Na+ of both electrodes. Linear plots of the data reveal a slope of about 48 mV for both electrodes. (C) Response of the V_Na+ of a double-barreled (black trace) and a concentric electrode (gray trace) to a fast change in the [Na⁺]_b from 152 mM to 70 mM. Note that the response time of the concentric electrode is significantly faster. For illustration purposes, traces were smoothed with a Sawitzky-Golay filter (width 20). Please click here to view a larger version of this figure.

9. Determine the response time of the electrodes, which depends on the tip diameter, the form of the tip as well as on the thickness of the sensor phase in the electrode tip.
   NOTE: In this experimental set up and employing a rapid change between different perfusion salines (exchange completed at the electrode’s tip within 500 msec), double-barreled sodium-sensitive microelectrodes reached a stable potential within 9.7 ± 4.5 sec when switching from 152 mM to 70 mM [Na⁺] (n=6). Within the same setting, concentric sodium-sensitive microelectrodes reached a stable potential within only 0.8 ± 0.3 sec (n=6) (Figure 6C). This emphasizes the superior time resolution of concentric as compared to double-barreled microelectrodes.

5. Dissection of Tissue

1. For generation of acute slices, anaesthetize mice of postnatal days 16-20 with CO₂ and quickly decapitated (following the recommendation of the European Commission published in: Euthanasia of experimental animals, Luxembourg: Office for Official Publications of the European Communities, 1997; ISBN 92–827-9694-9).
2. Prepare hippocampal tissue slices (thickness 250 µm), following a standard procedure (see e. g. ¹⁴).

6. Experimental Procedures

1. Transfer a slice into the experimental chamber and fix it with a grid.
2. Lower the calibrated ion-selective microelectrode onto the slice surface and impale the stratum radiatum of the CA1 region to a depth of about 50 µm with the electrode.
   NOTE: Touching the slice surface with the microelectrode leads to characteristic fluctuations in the ion-sensitive barrel. Furthermore, damage to cells during impalement of the tissue causes fluctuations as well. Wait until baseline has stabilized.
3. For pressure application of transmitter agonists, fill an application pipette with compound (e. g. 0.5 mM glutamate). Attach the pipette to a micromanipulator and couple it to a pressure application device.
4. Lower application pipette into the tissue and carefully place it in the proximity of the tip of the ion-selective microelectrode (~20-40 µm). Start pressure application (e. g. 10 sec, 40 mbar) and record voltage changes as monitored by the ion-selective microelectrode.
5. For bath application of drugs, switch between different perfusion salines for a defined duration.
6. To terminate the experiment, carefully withdraw the ion-selective microelectrode from the tissue and record any drift in potentials from the original zero value that might have occurred during prolonged recording periods.
7. Remove slice preparation from the bath and perform a second calibration of the ion-selective microelectrode.
   NOTE: This is necessary because the electrode tip can get clogged during its movement through the tissue. Thus, it is essential to ensure that the electrode still responds properly to changes in ion concentrations. Experiments should be rejected if the electrode’s response to a ten-fold change in ion concentration has dropped by more than 10%. Well-working electrodes can in principle be re-used in several experiments. This is especially true for concentric electrodes, which can even last for several days. It is essential, however, that calibration procedures are repeated before and after each single experiment to ensure the proper functioning of the electrodes.

7. Data Analysis

1. To convert the voltage response of the ion-selective electrode to changes in extracellular ion concentration, first convert equation 2 to determine the change in the potential ΔV_ion (mV) as follows (demonstrated here for K⁺-sensitive electrodes, analog procedure applies for Na⁺-sensitive electrodes):

V_K+: changes in the potential of the valinomycin barrel (mV)

s: Nernst slope

K⁺]_B: baseline concentration of potassium (in our case 2.5 mM K⁺)

K⁺]_o: changes in [K⁺]_o during experiment

2. Calculate the arithmetic average of the slopes derived from the single logarithmic plots of the calibration before and after the experiment (cf. Figure 5B, 6B; see point 4.8) to retrieve “s”.
3. To calculate changes the [K⁺]_o (Δ[K⁺]_o, in mM), rearrange equation 3 to: