Name: double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue
Uploaded: 2015-09-05T14:00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

The authors wish to thank C. Roderigo for expert technical assistance. We thank S. K&#246;hler (Center of Advanced Imaging, Heinrich Heine University Duesseldorf) for help in video production. Research in the author&#8217;s laboratory has been funded by the German Research Association (DFG: Ro 2327/8-1 to CRR), the Heinrich Heine University Duesseldorf (to NH) and by National Institutes of Health grant R01NS032123 (to MC).

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D., Heinemann, U., Dietzel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+changes+and+alterations+in+the+size+of+the+extracellular+space+during+epileptic+activity.">Ionic changes and alterations in the size of the extracellular space during epileptic activity.</a> Adv Neurol. 44, 619-639 (1986).</li><li>Zanotto, L., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartate+and+glutamate+induced+reductions+in+extracellular+free+calcium+and+sodium+concentration+in+area+CA1+of+'in+vitro'+hippocampal+slices+of+rats.">Aspartate and glutamate induced reductions in extracellular free calcium and sodium concentration in area CA1 of 'in vitro' hippocampal slices of rats.</a> Neurosci Lett. 35, 79-84 (1983).</li><li>Borrelli, M. J., Carlini, W. G., Dewey, W. C., Ransom, B. 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The authors declare no competing interests.

Liquid-carrier-based, ion-selective electrodes have been successfully employed for decades and for many ions, highly specific sensors are available 22-26. When used in the extracellular space (ECS) of vertebrate brain preparations, one must keep in mind, however, that this is a quite invasive technique: while the width of the ECS is only around 20-50 nm, the diameter of ion-selective microelectrodes is about 1 &#181;m (double-barreled electrodes) or larger (concentric electrodes). The tips of ion-selective microelectrodes will thus not only damage tissue during their impalement of the tissue, but also enlarge the ECS, favouring an underestimation of ion transients. Despite these pitfalls, extracellular ion transients in response to neuronal activity are remarkably consistent between different laboratories 7,8, attesting to the reliability of this method.
The performance and suitability of ion-selective electrodes is dependent on their sensitivity and selectivity, which is defined by the sensor cocktail (&#39;liquid membrane ionophore&#39;) used. Sensor cocktails contain a special carrier molecule, e.g. valinomycin for K+-selective microelectrodes which exhibits a high selectivity for potassium 27. Notwithstanding, cross-reactivity with other ions can occur and must be tested. Valinomycin exhibits a significant cross-reactivity for ammonium, which has to be considered when interpreting results (e.g. 11,12). Furthermore, because the voltage-response of the ionophores follows a Nernstian behavior (cf. equation 1), the signal-to-noise ratio and detection threshold depend on the concentration of the ion to be measured. Thus, while small [K+]o transients evoke large voltage changes against the low baseline [K+]o, small [Na+]o transients are much more difficult to detect against the high baseline [Na+]o (cf. Figure 5 and 6).
The performance of ion-selective electrodes is also determined by the temporal resolution, which is largely governed by its electrical time constant. The latter is mainly determined by the axial resistance of the sensor, and by the distributed capacitance along the length of the pipette, between its internal solutions and the external fluid. In the double-barreled configuration, the resistance is high, owing to the long column of backfilled ion sensor. For a given insulating dielectric (in this case borosilicate glass), the capacitance is governed by the dielectric thickness. In double-barreled electrodes, the dielectric width amounts to the glass wall of the pipette. As the glass thins close to the tip, the dielectric width falls, and the capacitance increases. These factors combine to produce electrodes with response times that range from several hundred milliseconds to several seconds, as these factors are varied.
A major advantage of the concentric design is that both the axial resistance and the capacitance to the bath are greatly diminished. The concentric pipette shunts most of the resistance of the backfilled ion exchanger, leaving only a remnant in the last few micrometers before the tip. In addition, the filling solution within the concentric pipette is physically distanced from the bath, separated by the thickness of two glass walls, greatly reducing the capacitance. As shown earlier 10, the combined effect of reduced resistance and capacitance is an improvement in temporal resolution of two orders of magnitude. In the case of concentric Ca2+ and pH microelectrodes, 90% response times were as low as 10-20 msec 10. A related advantage of the concentric design is the lower noise level (cf. Figure 8). Owing to the greatly reduced resistance, voltage transients from any ambient noise are minimized. Moreover, recovery from such transients is rapid, because of the fast time constant. Such artifacts are therefore small and fast, and have a less disruptive effect on physiological recordings (cf. Figure 8).
There are also disadvantages of the concentric technique. First, their assembly is more complex, and time-consuming. A second disadvantage is the need to place a separate reference microelectrode with its tip, entailing use of either a separate micromanipulator or a specialized dual manipulator. Finally, double-barreled microelectrodes can be extended to a triple-barreled design, allowing detection of two different ion species at the same time 28, which is not possible for concentric electrodes.
Most common pitfalls
Inefficient silanization.
The most important step, and principal obstacle in fabrication of any liquid-sensor based ion-selective microelectrode is the silanization procedure. When electrodes fail to respond to changes in specific ion concentration, or respond with a sub-Nernstian response (i.e., well less than 58 mV per ten-fold concentration difference), poor efficacy of silanization is typically the cause. In our experience, this can occur if atmospheric humidity is too high, or too low, typical of conditions at the height of summer, or winter, respectively. If it is feasible to exert some control over room humidity, these problems may be overcome.
Electrode resistance is too high.
If needed, the resistance of the ion-sensitive barrel can be reduced by bevelling. To this end, expose its tip to a strong jet of an abrasive suspended in water for a couple of seconds. This will cause its upmost tip to break and lower the resistance to the desired value.
Salt bridges.
Salt bridges between the ion and reference barrels result in poorly or none-responding electrodes and can thus also greatly confound their performance in the calibration. As mentioned above (see point 1.6.), this is mainly an issue when double-barreled theta glass is chosen, but is a rare occurrence when using the offset, twisted barrel technique described here.
With ease of fabrication in mind, the original double-barreled design of Lux 29 can often be used profitably. This method utilizes pre-filling of the ion and reference barrels with salt solutions, a fast exposure to a silane solution by its suction and expulsion from the tip, following by incorporation of ion-exchanger, also via the tip (see 30,31). These electrodes can be fabricated in roughly 10 min, but their tip size is typically 4 &#181;m or more and they are more prone to fail during an experiment. In contrast, silanization methods that involve exposure to silane vapor and heating can produce electrodes with smaller tips that last days, and sometimes weeks.
Taken together, there are several protocols and approaches on how to prepare ion-selective microelectrodes. Here, we have described two main procedures for fabrication of twisted double-barreled as well as concentric microelectrodes which work well and reliably in our laboratories, with an overall success rate of close to 100%. Importantly, these techniques will be transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations than the brain, including fluid-filled cavities or fluids in general. Last, but not least, ion-selective microelectrodes allow determination of ion concentrations inside cells. Because of their relatively large tip size (~ 1 &#181;m), this will, however, be possible only in cells with a large cell body, e.g. such as found in invertebrate preparations 28,32.

An author's middle initial was omitted from the publication, <a href="http://www.jove.com/video/53058/double-barreled-concentric-microelectrodes-for-measurement">Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue</a>. The author&#39;s name has been updated to:
Christine R. Rose
from:
Christine Rose

A correction was made to Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue.

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

Electrical signaling in the brain is based on the flux of ions across plasma membranes. Major ion movements into and from the extracellular space are not only mediated by passage through voltage-gated ion channels, but also by postsynaptic ionotropic receptors as well as ion transporters. Neuronal activity is thus accompanied by complex changes in the extracellular concentration of all major ions 1. For example, influx of sodium into neurons during excitatory activity has been shown to result in a decrease in the extracellular sodium concentration ([Na+]o) 2. The same holds true for the extracellular calcium concentration because calcium ions rapidly enter both pre- and postsynaptic structures 3. At the same time, potassium moves the opposite way and this mediates an increase in the extracellular potassium concentration ([K+]o) in the low mM range 4,5. Synaptic activity also causes changes in extracellular pH that are partly mitigated by concomitant glial membrane fluxes that change intraglial pH 6,7. These activity-related changes in extracellular ion concentrations have significant functional consequences. For example, even small increases in [K+]o depolarize neurons as well as glial cells thereby altering neuronal excitability, and several mechanisms exist to remove excess potassium 8. Failure of these may result in epileptiform activity of neurons or phenomena like spreading depression 1. 
Because of their critical importance, quantitative determination of extracellular ion concentrations is often necessary and required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. For decades, double-barreled ion-selective microelectrodes have proven to be excellent tools for such measurements in brain tissue 9. For many ions, highly specific sensors with low cross-reactivity for other ions are available. In addition to the classical double-barreled electrodes, so-called concentric electrodes were recently introduced. The latter provide a superior time resolution, but take a little more time and effort to construct 10. 
In the following, we will describe the preparation and calibration of these two types of ion-selective microelectrodes. We then show how these electrodes can be employed in brain slice preparations for measurement of changes in [K+]o or [Na+]o induced by excitatory activity following different stimulation paradigms including bath and pressure application of drugs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Abrasive&#160;&#160;</td>
			<td>MicroPolish&#160;</td>
			<td>Buehler GmbH</td>
			<td>Dissolved in A.dest</td>
		</tr>
		<tr>
			<td>Borosilicate-glass capillaries</td>
			<td>1405059</td>
			<td>Hilgenberg</td>
			<td>Application pipette; 75 mm x 2 mm, wall thickness 0.3 mm</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC 150 F-15&#160;</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the sensor of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GC100-F-15</td>
			<td>Clark Electromedical Instruments, Harvard Apparatus</td>
			<td>For the reference of double-barreled microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-200TF-15&#160;</td>
			<td>Science Products</td>
			<td>Concentric, outer channel. o.d. 2.0 mm&#160;&#160;</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries with filament</td>
			<td>GB-120TF-10</td>
			<td>Science Products</td>
			<td>Concentric, inner channel. o.d. 1.2 mm</td>
		</tr>
		<tr>
			<td>Digidata</td>
			<td>1322A</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrometer amplifier with headstage</td>
			<td></td>
			<td>Custom-made</td>
			<td>Rin = 10T&#937; and Ibias=50fA-1pA (commercially available&#160; alternatives: e. g. Dagan IX2-700, with headstage (10 Gig feedback resistor) or EPMS-07, NPI, Tamm, Germany)</td>
		</tr>
		<tr>
			<td>Experimental chamber</td>
			<td></td>
			<td>Custom-made</td>
			<td>Commercially available from e.g.&#160; Warner Instruments,USA;&#160; Scientifica, UK</td>
		</tr>
		<tr>
			<td>Furnace</td>
			<td></td>
			<td>Heraeus</td>
			<td>Must stay constant at 200&#176;C</td>
		</tr>
		<tr>
			<td>Hard sticky wax / dental wax&#160;</td>
			<td>Deiberit 502&#160;</td>
			<td>Siladent Dr. Boehme &#38; Schoeps GmbH</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td></td>
			<td>Custom-made</td>
			<td>Must stay constant at 40&#176;C</td>
		</tr>
		<tr>
			<td>Microelectrodes holder made of plexiglas</td>
			<td></td>
			<td>Custom-made</td>
			<td>Double-barreled: o.d.&#160; capillaries 1.5 mm, concentric: o.d.&#160; capillaries 2 mm</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td></td>
			<td>Leitz</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>MD4R</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>M205C</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Plan 0.8xLWD</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model PP-830</td>
			<td>Narishige</td>
			<td>Concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Model P-97</td>
			<td>Sutter Instruments&#160;</td>
			<td>Sensor of concentric microelectrodes&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic drug ejection system</td>
			<td>Picospritzter Type II</td>
			<td>General Valve TM Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>Travel dovetail stage</td>
			<td>DT 25/M</td>
			<td>Thorlabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-component glue</td>
			<td>&#160;Araldite</td>
			<td>Huntsman advanced materials GmbH</td>
			<td>One may also use a small stripe of aluminum foil to stick the capillaries together&#160;</td>
		</tr>
		<tr>
			<td>Silverwire</td>
			<td>99.9%&#160;</td>
			<td>Wieland Edelmetalle</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer / Vibratome</td>
			<td>Microm HM 650 V</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>AxoScope 8.1&#160;</td>
			<td>Axon Instruments</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical puller&#160;</td>
			<td>Type PE-2</td>
			<td>Narishige Scientific Instruments</td>
			<td>With a revolvable chuck for&#160; double-barreled microelectrodes</td>
		</tr>
		<tr>
			<td>x/y translational stage</td>
			<td></td>
			<td>Custom-made</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Compound</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>1(S),9(R)-(&#8722;)-Bicuculline methiodide</td>
			<td>Sigma aldrich</td>
			<td>14343</td>
			<td>Competitive antagonist of GABAA receptors (light-sensitive); CAUTION toxic</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma aldrich</td>
			<td>C-127</td>
			<td>AMPA/kainate receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-AP5</td>
			<td>Alfa Aesar</td>
			<td>J64210</td>
			<td>NMDA receptor antagonist; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma aldrich</td>
			<td>440191</td>
			<td>CAUTION: Flammable, acute toxicity (oral, dermal, inhalation) and corrosive to metals and skin</td>
		</tr>
		<tr>
			<td>L-Aspartic acid</td>
			<td>Sigma aldrich</td>
			<td>A9256</td>
			<td>Activates NMDA and non-NMDA and EAATs</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma aldrich</td>
			<td>G1626</td>
			<td>Activates NMDA-R, AMPA-R, QA-R and KA-R),&#160; mGluRs and EAATs</td>
		</tr>
		<tr>
			<td>Potassium ionophore I - cocktail B</td>
			<td>Fluka&#160;</td>
			<td>60403</td>
			<td>Based on valinomycin; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Sodium ionophore II - cocktail A</td>
			<td>Fluka&#160;&#160;</td>
			<td>71178</td>
			<td>Based on ETH 157</td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Ascent Scientific</td>
			<td>Asc-055</td>
			<td>Inhibitor of voltage-dependent Na+ channels; CAUTION toxic</td>
		</tr>
		<tr>
			<td>Water, ultra pure</td>
			<td>Sigma aldrich</td>
			<td>W3500&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

double-barreled and concentric microelectrodes for measurement of extracellular ion signals in brain tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

To monitor [K+]o and changes therein in response to excitatory activity, a double-barreled potassium-selective microelectrode was inserted into the stratum radiatum of the CA1 area. A few minutes after impalement, the ion-selective barrel of the electrode reached a stable baseline (Figure 7A), corresponding to a [K+]o of about 2.8 mM, a value close to the [K+] of the ACSF used (2.5 mM). Bath application of 0.5 mM glutamate for 10 sec induced a reversible increase in [K+]o by about 4 mM, followed by an undershoot below baseline amounting to ~0.7 mM (Figure 7A). Lowering the glutamate concentration to 0.4, 0.3, and 0.2 mM caused a corresponding reduction in the amplitude of both the transient increase and the undershoot in [K+]o (Figure 7A).
<img alt="Figure 7" src="/files/ftp_upload/53058/53058fig7.jpg" /> 
Figure 7. Extracellular potassium transients inresponse to excitatory activity. (A) [K+]o transients, consisting of an increase followed by an undershoot below baseline, induced by bath application of different concentrations of glutamate for 10 sec as indicated. (B) Influence of a perfusion with glutamate receptor blockers (CNQX, 100 &#181;M; APV, 100 &#181;M) and tetrodotoxin (TTX; 0.5 &#181;M) on [K+]o transients induced by bath application of 0.5 mM glutamate for 10 sec. (C) Spontaneous [K+]o transients in the presence of 0Mg2+/BIC. Experiments shown in A-C were performed using double-barreled electrodes. For illustration purposes, traces were smoothed with a Sawitzky-Golay filter (width 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Earlier experiments have shown that such glutamate-induced [K+]o signals are not significantly altered during application of TTX, and are thus largely independent on the opening of voltage-gated sodium channels and neuronal action potential generation 15,16. To study the mechanisms underlying the observed [K+]o changes in response to glutamate, we applied glutamate receptor blockers, namely CNQX (100 &#181;M; blocker of AMPA receptors) and APV (100 &#181;M; blocker of NMDA receptors) in the presence of TTX (0.5 &#181;M). Upon bath perfusion with these blockers, glutamate-induced [K+]o changes were virtually abolished, confirming their dependence on the opening of ionotropic glutamate receptor channels as reported before (e.g. 17; Figure 7B).
To further demonstrate the relevance of glutamatergic excitation for the generation of extracellular [K+] signals, slices were perfused with nominally Mg2+-free saline containing 10 &#181;M bicuculline methiodide (0Mg2+/BIC). This relieves voltage-dependent Mg2+-block of NMDA receptors and dampens inhibition by blocking GABAA receptors, causing spontaneous recurrent epileptiform activity in the network (e.g. 18,19). As expected 20, spontaneous, recurrent [K+]o transients, amounting to about 1.5 mM were detected in 0Mg2+/BIC saline (Figure 7C). In between these responses, smaller [K+]o transients with a mean amplitude of about 0.2 mM occurred (Figure 7C).
In a second set of experiments, we used [Na+]-sensitive microelectrodes to determine [Na+]o changes evoked by application of glutamate agonists. Here, we also compared the response characteristics of double-barreled and concentric microelectrodes employing two different application paradigms. [Na+]-sensitive microelectrodes were positioned into the stratum radiatum at a depth of about 50 &#181;M. After a stable baseline was attained, the glutamate agonist L-aspartate was applied per bath perfusion (10 mM, 120 sec, bath perfusion at 2.5 ml/min). As observed earlier 21, application of aspartate caused a slow decrease in [Na+]o by roughly 15 mM, which lasted about 5 min and then started to recover back to baseline (Figure 8A). Notably, while the peak amplitudes and kinetics of the [Na+]o signals determined by both electrodes were virtually identical under these conditions, concentric electrodes exhibited a more stable baseline and a lower noise level (Figure 8A).
To test the response characteristics of the different electrodes under a more rapid application paradigm, we pressure-applied glutamate through a fine glass pipette positioned in the stratum radiatum at a distance of 20-40 &#181;m from the tip of the ion-selective microelectrode. As expected 21, application of glutamate (10 mM) for 200 ms caused a drop in the [Na+]o (Figure 8B). The peak amplitudes of the [Na+]o decrease were in the same range for both types of electrodes (double barreled: 4.5 - 13.5 mM, n=14; concentric: 2.0 - 19.1 mM, n=15). However, in contrast to the results obtained with slow bath perfusion (see above), not only the signal-to-noise ratio, but also the time course of the [Na+]o signals detected by the two types of electrodes differed significantly. The average time to peak was 3.5 sec for the double-barreled and only 1.3 sec for the concentric microelectrodes.
Thus, these results demonstrate and confirm the faster response kinetics of concentric vs. double-barreled Na+-selective microelectrodes, (cf. Figure 6C and 8B), as was also noted for Ca2+ and pH-selective counterparts 10. In contrast to the former study, in which short-burst synaptic stimulation was performed to evoke fast synaptically-induced ion transients, there was only a tendency, but no significant difference in mean peak amplitudes between concentric and double-barreled electrodes with our application paradigm. This was probably due to the fact that the distance of the application pipette from the tip of the ion-selective microelectrode varied by a factor of two (20-40 &#181;m), and consequently, that the maximal glutamate concentration at the target region was not the same in different experiments, obstructing any existing difference in peak amplitudes.
(A) Top traces: Change in the voltage of the reference barrels (Vref) 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 (VNa+) 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 VNa+ of both electrodes. Linear plots of the data reveal a slope of about 48 mV for both electrodes. (C) Response of the VNa+ 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).
<img alt="Figure 8" src="/files/ftp_upload/53058/53058fig8.jpg" /> 
Figure 8: Extracellular sodium transients as detected by double-barreled and concentric electrodes. (A) Transient changes in Vref and [Na+]o induced by bath perfusion with 10 mM aspartate for 120 sec as indicated by the bar. The upper traces show a recording performed with a double-barreled electrode, the lower traces were recorded using a concentric electrode. (B) Transient changes in Vref and [Na+]o induced by local pressure application with 10 mM glutamate for 0.2 sec as indicated by the arrowhead. The upper traces show a recording performed with a double-barreled electrode, the lower traces were recorded using a concentric electrode. Dotted red lines represent linear fits of the period between the start of the signal to its maximum. Note that the response time of the concentric electrode is significantly faster under this condition. For illustration purposes, traces were smoothed with a Sawitzky-Golay filter (width 20). <a href="https://www.jove.com/files/ftp_upload/53058/53058fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue at JoVE.com

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

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

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