Potassium ions contribute 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 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 ion concentrations are tightly regulated in the brain, and their fluctuations exert a powerful influence on the resting membrane potential of all cells. In light of these critical contributions, an important goal of biology is to determine the cellular and biophysical mechanisms that are used to tightly regulate the concentration of K+ in the extracellular space in different organs of the body1,2. An important requirement in these studies is the ability to measure K+ concentrations accurately. Although many components which contribute to potassium homeostasis in the brain in healthy and diseased states have been identified3,4,5, further progress has been slowed due to the specialized nature of preparing ion selective microelectrodes for potassium measurement. Microelectrode sensors represent the gold standard for measuring K+ concentrations in vitro, in tissue slices and in vivo.
Newer approaches for K+ monitoring are under development using optical sensors, however these do not detect a biologically relevant range of K+ concentrations or have not been fully vetted in biological systems, although initial results appear promising6,7,8. Compared to optical sensors, microelectrodes are fundamentally limited to a point source measurement of ions, although electrode arrays could improve the spatial resolution9. This article focuses on the single-barreled microelectrode sensors for monitoring K+ dynamics.
In this work, we report detailed stepwise procedures to make K+ selective microelectrodes, using a valinomycin-based potassium ionophore that permits highly selective (104 fold K+ to Na+ selectivity) K+ movement over membranes10. A naturally occurring polypeptide, valinomycin acts as a K+ permeable pore and facilitates the flow of K+ down it's electrochemical gradient. We also describe how to calibrate the electrodes, how to store and use them and finally how to deploy them to measure K+ concentration dynamics in acute hippocampal brain slices from adult mice. The use of such electrodes together with genetically modified mice that lack specific ion channels proposed to regulate extracellular K+ dynamics should reveal the cellular mechanisms used by the nervous system to control the ambient concentration of K+ in the extracellular milieu.
All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Chancellor's Animal Research Committee at the University of California, Los Angeles. All mice were housed with food and water available ad libitum in a 12 h light-dark environment. All animals were healthy with no obvious behavioral changes, were not involved in previous studies, and were sacrificed during the light cycle. Data for experiments were collected from adult mice (6-8 weeks old for all experiments).
1. Preparation of K+ selective microelectrodes
2. Calibration of K+ Selective Microelectrodes
Chemical | MW | final mM | 0.1 mM [K+] | 1 mM [K+] | 4.5 mM [K+] | 10 mM [K+] | 100 mM [K+] |
(g / mol) | |||||||
NaCl | 58.44 | varies | 1.51 g | 1.50 g | 1.44 g | 1.4 g | 0.345 g |
KCl | 1 M stock | varies | 20 µl | 200 µl | 900 µl | 2 ml | 20 ml |
CaCl2 | 1 M stock | 2 | 400 µl | ||||
MgCl2 | 1 M stock | 1 | 200 µl | ||||
NaH2PO4 | 119.98 | 1.2 | 0.29 g | ||||
NaHCO3 | 84.01 | 26 | 0.437 g | ||||
D-Glucose | 180.16 | 10 | 0.360 g | ||||
Water | q.s. 200 ml |
Table 1. Potassium calibration solutions
3. Preparation of Acute Hippocampal Brain Slices
4. Measurement of Electrically Evoked K+ Dynamics
For selective measurement of extracellular K+, we prepared ion-selective microelectrodes coated with a hydrophobic layer through silanization of clean borosilicate glass pipettes (Figure 1A). This coating enables the K+ ionophore containing valinomycin to rest at the tip of the electrode and permit only K+ flux through a narrow opening at the electrode tip (Figure 1B). After priming the electrodes with the backfilled saline solution and the K+ ionophore, the electrodes can be tested for their rapid and linear response to stepwise changes in bath K+ concentrations (Figure 3A) and for their response to bath K+ changes over the calibration range (Figure 3B) in saline or ACSF in a manner predicted by the Nernst equation2. The change in the steady state potential can be plotted against the bath K+ concentration in order to determine the slope of the line, which should approximately be 58.2 mV per log [K+], according to the Nernst equation, and no less than 52 mV per log [K+] (Figure 3C). We additionally tested the responsiveness of the K+ selective electrodes and found that they responded to a 5.5 mM change in K+ with rise and decay time constants of approximately 85 ms (Figure 3D,E).
The electrophysiological recording rig consists of a standard upright microscope connected to an LCD display for identifying the placement of the stimulating and recording electrodes. No special optics are needed for visual placing of the K+ and stimulation electrodes; we use a 5x or 10x objective lens and white light from a halogen bulb, but a white LED could be used instead. The stimulating electrode is connected to the output of a stimulus isolator, which delivers depolarizing current via the timed delivery of pulses from a stimulator or other such timing device. In other words, the stimulator delivers trains of 2 V, 10-20 Hz timing pulses to the stimulus isolator. Upon receiving these pulses, the stimulus isolator then delivers the desired current to the stimulation electrodes. The recording electrode is connected to an electrode holder, connected to a headstage, amplifier and A/D board, which interface to a PC with electrophysiological recording software (Figure 4A). After the electrode has been successfully calibrated and the acute slices have been prepared, the slice can be placed in the ACSF perfusate. To stimulate the Schaffer collaterals, the K+-selective electrode is placed within the CA1 stratum radiatum and the field stimulation electrode is placed within the CA3 (Figure 4B).
Once the electrodes have been placed and the K+ recording has reached a stable baseline, then pulses of increasing current amplitude can be applied to the slice (Figure 5, top). The waveform of this activity appears as a rapid increase in K+ with an exponential decay rate, which is abolished with TTX application (Figure 5, bottom).
Figure 1: Diagram of silanization reaction and K+ selective microelectrode architecture. A. Schematic representation of silanization reaction that occurs between the exposed polar hydroxyl groups of the borosilicate glass and the silanization reagent dichlorodimethylsilane (DDS). This reaction renders the surface of the glass hydrophobic, which allows the K+ ionophore to form a thin membrane. B. Diagram of the K+ selective microelectrode. The electrode is backfilled with saline solution and the K+ selective solution is place in a 1-2 mm thick layer at the tip. Please click here to view a larger version of this figure.
Figure 2: Diagram of DDS extraction. Schematic representation of the nitrogen replacement procedure for DDS extraction from a container. DDS is volatile and flammable and can react violently with atmospheric gases when it is in high concentrations therefore it is necessary to replace the DDS removed with inert nitrogen gas. A balloon filled with nitrogen is connected to a needle via a syringe or appropriate tubing. This needle is inserted through the sealant on the container allowing nitrogen gas (N2) flow into the container. Separately, a long (3-10 cm) needle is connected to a 1 mL syringe and inserted into the container. This syringe is then used to extract DDS, while only nitrogen gas can enter the container. Please click here to view a larger version of this figure.
Figure 3: Calibration of microelectrodes. A. Bath perfusion application of different K+ concentrations in saline can rapidly and reversibly produce Nernstian changes in the potential across the electrode tip. B. Stepwise application of K+ in ACSF evokes a characteristic and stable change in the electrode tip potential. C. Plot of the mV change of the K+ selective electrode in response to increasing concentrations of K+ in four electrodes; the R2 is 0.9995 for these four electrodes. D. Fast perfusion system bath application of 10 mM K+ causes a step response in voltage across the K+ selective electrode tip. E. Plot of the measured response times (tau in ms); no difference between rise and decay time was detected (mean ± SEM, p = 0.939, two sample t-test). Please click here to view a larger version of this figure.
Figure 4: Apparatus for extracellular K+ measurement. A. The electrophysiology rig consists of a microscope fixed to an anti-vibration table with an attached camera and LCD display for slice visualization. The K+ electrode is fixed to a headstage, amplifier and analog to digital board which outputs the signal to an attached PC with electrophysiological recording software. The electrical stimulation electrodes are connected to a stimulus isolator which varies the stimulation amplitude and a stimulator for timing stimulus delivery. B. Diagram of the slice preparation and the location of the placement of the various electrodes at the slice. CA3 can be approximately identified as the portion of the hippocampus proper lateral to the granule cell layer at the hippocampal genu, with the stratum radiatum falling rostral to the pyramidal cell layer. Please click here to view a larger version of this figure.
Figure 5: Measurement of electrically evoked K+ release. Representative traces of K+ release from the recording electrode under basal conditions (top) and their loss upon the application of tetrodotoxin (0.25 µM) for 5 minutes prior to recording (bottom). Please click here to view a larger version of this figure.
The method that we describe here has allowed us to assess K+ dynamics in response to electrical stimulation of Schaffer collaterals in acute hippocampal slices from adult mice. Our method of preparing K+ ion selective microelectrodes is similar to earlier described procedures12,13,14,15. However, this method has advantages over alternative electrode configurations in that it is rapid and uncomplicated to prepare K+ selective microelectrodes. After appropriate calibration, these electrodes were found to robustly measure K+ dynamics in slices during electrical stimulation, and such responses were blocked by TTX. In these experiments, stimulations of 80-160 uA at 10 Hz were used; however, optimization of stimulation conditions for a particular experiment and for a brain area of interest will be required. These values are listed as a guide.
The slope of the response of the electrodes should be 58.2 mV per log [K+]. Such a value is predicted from the Nernst and Nicolsky-Eisenman equations for a K+ selective semi-permeable membrane; the latter better accounts for interactions between ions16. If the electrode does not respond in the predicted manner, this could be for one of two primary reasons. First the silanization could be inadequate, causing the membrane to be lost or salt bridges to form. Confirm that the membrane is intact by observation through the microscope, there should be a clear interface between the pipette solution and the membrane. Another reason, could be the presence of bubbles in the pipette that impede the flow of current from the silver chloride wire. If bubbles are observed, then remove the pipette and flick vigorously to remove them. If these solutions fail, re-make another K+ selective electrode or repeat silanization for longer or at higher temperatures. However, it is important to repeat these key controls every time a new brain region is studied or a new microelectrode is tested.
Two specific amplifiers were used in these experiments, but other amplifiers could be used so long as the input impedance is greater than or equal to 500 MΩ. Having calibrated the electrodes with both 500 MΩ and 5 GΩ input impedances, we found there was no difference in the slope of the voltage response with either setting over a range of K+ concentrations (56.9 ± 0.7 and 56.5 ± 0.9 mV per ten-fold change in [K+] for 500 MΩ and 5 GΩ input impedances, respectively; P = 0.759, paired t-test, n = 4 electrodes).
We also used fast solution switches to estimate the response time of the electrodes to a known jump in [K+] from 4.5 to 10 mM (Figure 3D). The electrodes responded with rise and decay times (tau) of 85 ± 12 and 85 ± 15 ms, respectively. In relation to this, the solution exchange kinetics for our custom fast solution switcher was 85 ± 27 ms. Hence, the K+ selective electrodes respond with kinetics as fast as the solution switcher we employed and far faster than the dynamics of K+ in the extracellular milieu as a result of Schaffer collateral stimulation (Figure 5). These data suggest that K+ selective electrodes can be used to estimate the kinetics of K+ accumulation and clearance in brain tissue. However, in future work appropriate controls and calibrations will be needed on a case-by-case basis. We suggest that it is valuable to spend time understanding the solution exchange kinetics in your recording chamber.
We found three critically important factors that influence the quality and robustness of K+ measurements in slices. The first is the quality of the preparation, both the tissue health and age of the animals used appear to be relevant. For this study, we have used C57/Bl6N mice that are ~12 weeks old. On rare occasions, we found slices with spontaneous K+ fluctuations amounting to ~0.1 mM and lasting 5-10 seconds; these slices were discarded. The second is the quality of the recording microelectrode. The primary issue is the time and temperature that is used for silanization of the pulled glass capillary. We recommend >170 °C for at least 6 hours (up to overnight is acceptable) or at 200 °C for 30 minutes. Insufficient heating of the electrodes with the silanization reagent can lead to electrodes that do not maintain a steady state potential because of gradual loss of the K+ ionophore. Additionally, when preparing the electrodes, we recommend placing a thin layer (1-2 mm) of the K+ ionophore in the tip with a diameter of 10-20 µm i.e. at approximately the size of an average cell body (Figure 1B). Do not break the tip excessively wide, or the K+ selective membrane will lose integrity and the electrode will fail. This step may require some practice to achieve tips of the correct size. K+ selective electrodes with a too fine tip or too thick of a layer can have sluggish responses, compared to properly constructed electrodes. The third factor is the distance between the stimulation electrode and the K+ selective electrode. We have used an inter-electrode distance of approximately 500 µm, however the optimal distance can vary considerably with the individual brain area and will need to be carefully considered for the particular experiment at hand.
In addition to the single barrel configuration, there are currently several methods for making K+-selective microelectrodes in bipolar and concentric formats17. Compared to the published descriptions of these methods, single channel electrodes have two main disadvantages: 1) a slightly larger tip diameter (~10 vs 4 µm), which could cause greater disruption of the extracellular space comparted to bipolar and concentric electrodes, and 2) incompatibility with simultaneous measurement of multiple ion species as in the bipolar electrodes. However, the single channel electrodes offer several advantages. Specifically, these electrodes can be fabricated in less than five minutes and are therefore more disposable and can be made and calibrated quickly before experiments. Therefore, the risk of electrode breakage during the course experiments is less of a concern. Additionally, because the ground electrode and recording electrode are physically separated by the volume of the bath, there is no chance for the formation of salt bridges at the tip of the electrode, which can lead to electrode failure in concentric and bipolar electrodes. The response time of the single channel electrodes is faster than bipolar electrodes and likely comparable to concentric electrodes (~20 ms), although our fast perfusion system only permitted a measurement of response times on the order of 80 ms (Figure 3E). Furthermore, these electrodes offer lower noise compared to bipolar electrodes, which have greater tip resistance and require the use of amplifiers with higher input resistance. Lastly, these electrodes do not require the use of a specialized micromanipulator or headstage as is required for concentric electrodes. On balance, the advantages of the construction and ease of use of single channel electrodes outweighs the disadvantages.
The approach that we used here to measure K+ dynamics in slices can be used in many brain regions to study K+ regulation. Although this protocol demonstrates the usage of K+-selective electrodes for the measurements of the dynamics of electrically evoked potassium ions in brain tissues, this protocol can be broadly used in many different tissues where it is desirable to measure K+ dynamics. Such situations may include spontaneous dynamics and changes in response to pharmacological, optogenetic, or chemogenetic cellular activation. These microelectrodes can be made with sufficient quality and reliability as to permit rapid integration of this technique into any laboratory toolbox. The detailed analyses of K+ concentrations in health and diseased states will enable further detection and quantification of how various molecular and cellular components contribute to resting K+ concentrations in the brain3,18,19.
The authors have nothing to disclose.
The Khakh lab was supported by NIH MH104069. The Mody lab was supported by NIH NS030549. J.C.O. thanks the NIH T32 Neural Microcircuits Training Grant(NS058280).
Vibratome | DSK | Microslicer Zero 1 | |
Mouse: C57BL/6NTac inbred mice | Taconic | Stock#B6 | |
Microscope | Olympus | BX51 | |
Electrode puller | Sutter | P-97 | |
Ag/AgCl ground pellet | WPI | EP2 | |
pCLAMP10.3 | Molecular Devices | n/a | |
Custom microfil 28G tip | World precision instruments | CMF28G | |
Tungsten Rod | A-M Systems | 716000 | |
Bipolar stimulating electrodes | FHC | MX21XEW(T01) | |
Stimulus isolator | World precision instruments | A365 | |
Grass S88 Stimulator | Grass Instruments Company | S88 | |
Borosilicate glass pipettes | World precision instruments | 1B150-4 | |
A to D board | Digidata 1322A | Axon Instruments | |
Signal Amplifier | Multiclamp 700A or 700B | Axon Instruments | |
Headstage | CV-7B Cat 1 | Axon Instruments | |
Patch computer | Dell | n/a | |
Sodium Chloride | Sigma | S5886 | |
Potassium Chloride | Sigma | P3911 | |
HEPES | Sigma | H3375 | |
Sodium Bicarbonate | Sigma | S5761 | |
Sodium Phosphate Monobasic | Sigma | S0751 | |
D-glucose | Sigma | G7528 | |
Calcium Chloride | Sigma | 21108 | |
Magnesium Chloride | Sigma | M8266 | |
valinomycin | Sigma | V0627-10mg | |
1,2-dimethyl-3-nitrobenzene | Sigma | 40870-25ml | |
Potassium tetrakis (4-chlorophenyl)borate | Sigma | 60591-100mg | |
5% dimethyldichlorosilane in heptane | Sigma | 85126-5ml | |
TTX | Cayman Chemical Company | 14964 | |
Hydrochloric acid | Sigma | H1758-500mL | |
Sucrose | Sigma | S9378-5kg | |
Pipette Micromanipulator | Sutter | MP-285 / ROE-200 / MPC-200 | |
Objective lens | Olympus | PlanAPO 10xW |