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 ...

double-barreled-concentric-microelectrodes-for-measurement

Research

JoVE Journal

Neuroscience

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K+ affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K+ dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K+-selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K+ concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K+ concentrations in the nervous system.

Potassium ions contribute&#160;to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K+ concentration is a crucial regulator ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural synchronization (INS) during social interactions, functional near-infrared spectroscopy (fNIRS) is one of the most popular techniques for hyperscanning naturalistic social interactions because of its relatively high spatial resolution, sound anatomical localization, and exceptionally high tolerance of motion artifacts. Previous fNIRS-based hyperscanning studies usually calculate a time-lagged INS using wavelet transform coherence (WTC) to describe the direction and temporal pattern of information flow between individuals. However, the results of this method might be confounded by the autocorrelation effect of the fNIRS signal of each individual. For addressing this issue, a method termed partial wavelet transform coherence (pWTC) was introduced, which aimed to remove the autocorrelation effect and maintain the high temporal-spectrum resolution of the fNIRS signal. In this study, a simulation experiment was performed first to show the effectiveness of the pWTC in removing the impact of autocorrelation on INS. Then, step-by-step guidance was offered on the operation of the pWTC based on the fNIRS dataset from a social interaction experiment. Additionally, a comparison between the pWTC method and the traditional WTC method and that between the pWTC method and the Granger causality (GC) method was drawn. The results showed that pWTC could be used to determine the INS difference between different experimental conditions and INS's directional and temporal pattern between individuals during naturalistic social interactions. Moreover, it provides better temporal and frequency resolution than the traditional WTC and better flexibility than the GC method. Thus, pWTC is a strong candidate for inferring the direction and temporal pattern of information flow between individuals during naturalistic social interactions.

This protocol describes partial wavelet transform coherence (pWTC) for calculating the time-lagged pattern of interpersonal neural synchronization (INS) to infer the direction and temporal pattern of information flow during social interaction. The effectiveness of pWTC in removing the confounds of signal autocorrelation on INS was proved by two experiments.

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...