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PubMed1993

Development of choline and acetylcholine Pt microelectrodes.

Choline (Ch) and acetylcholine (Ach) microenzyme sensors were developed based on the immobilization of choline oxidase (ChO) and acetylcholinesterase (AchE) at the tip of a 25-micron Pt wire sealed in glass. Several immobilization procedures were tested, including code-position of the enzyme/s with an electropolymer and cross-linking with glutaraldehyde. The various electropolymers used were 1,2-diaminobenzene, resorcinol, 4-hydroxybenzenesulfonic acid, and a combination of two polymers, 1,2-diaminobenzene and resorcinol. An inner membrane constructed from cellulose acetate (CA) was deposited prior to immobilization with glutaraldehyde. The analytical characteristics of the microelectrodes, including optimization of immobilization procedures, calibration curves, pH response curves, stability, and selectivity toward possible electroactive compounds found in the brain extracellular fluid, were determined. The best microelectrodes were prepared by cross-linking the enzymes with glutaraldehyde on top of the inner CA membrane. The responses are linear in the concentration range 5.0 x 10(-7)-1.0 x 10(-4) M Ch and 5.0 x 10(-7)-9.3 x 10(-5) M Ach. The time to reach 95% steady-state current was 15-20 s. The CA-coated Ch microelectrodes were useful for measurement of changes in Ch concentration in artificial brain extracellular fluid.

Acetylcholine↗

PubMed1996

Neutral carrier-based "Ca(2+)-selective" microelectrodes for the measurement of tetraphenylphosphonium.

Ca(2+)-selective microelectrodes with Simon's neutral carrier ETH 1001 are commercially available and have been widely used for the measurement of both extra- and intracellular calcium. The electrodes demonstrate high selectivity against other cations such as magnesium, sodium, and potassium. We report, however, that the ETH 1001-based microelectrode is a superior tetraphenylphosphonium (TPP+)-sensitive electrode. The electrode exhibits a Nernstian response for [Ca2+] > 10(-5) M but for [TTP+] > 10(-7) M. Using two different methods, we found that log kTTPCa (selectivity coefficient for TPP+ with respect to Ca2+) is in the range of -3.0 to -5.3. We argue that the ETH 1001 microelectrode can be used as a commercially available TPP+ electrode. We illustrate this application by making membrane potential recordings in respiring mitochondria. The results are identical to those obtained using conventional ion-exchange TPP+ electrodes.

Animals↗

PubMed1976

Measurement of current spread from microelectrodes when stimulating within the nervous system.

Tungsten stimulating microelectrodes have been tested in monopolar, bipolar and concentric configurations for the extent to which unwanted current spread occurred. Current spread from monopolar electrodes in close conformity with the predictions of the inverse square law, both in vitro in vivo. The bipolar and concentric configurations, tested only in vitro, had current-spread characteristics which did not follow the inverse square law so closely. The bipolar configuration gave little reduction in spread, compared wpith a monopolar electrode, but the concentric configuration did reduce the extent of stimulus spread. The extent of spread depends greatly upon a number of experimental variables, including the dimensions of the microelectrode tip. For studies requiring precise localization with low stimulus currents, it is advisable to determine the inverse square law constant for each experimental situation. For more generalized stimulation purposes within the mammalian C.N.S., employing monopolar microelectrodes of moderate tip size, brief pulses of 10 muA can be expected to stimulate myelinated axons with about 0.15 mm of the tip, and 100 muA WILL Stimulate with a radius of about 0.5 mm.

Animals↗

PubMed1988

Magnesium ion activity in the mammalian endolymph measured with ion-selective microelectrodes.

The free Mg++ concentration in endolymph was measured with Mg++-selective microelectrodes based on the neutral ligand ETH 1117. The property of Mg++ microelectrodes was obtained from calibration solutions, containing various Mg++ concentrations with the background electrolytes resembling endolymph. The range between 10 and 0.1 mM Mg++ concentrations changed the potentials of Mg++ microelectrodes by 14.4 +/- 3.0 mV. The endocochlear potential and the Mg++ concentration in the endolymph were 82.0 +/- 5.0 mV and 0.77 +/- 0.29 mM in the guinea pig, and 84.4 +/- 4.9 mV and 1.12 +/- 0.24 mM in the chinchilla, respectively. These results are discussed in the light of the dependence of Na+, K+-ATPase and its interaction with Ca++.

Animals↗

PubMed1988

A method for the manufacture of single barrel liquid ion-selective microelectrodes: an in situ study of ant venom pH.

A method for the manufacture of single barrel ion-sensitive microelectrodes, employing liquid ion-selective sensors, is described in detail. This method had proven reliable and repeatable for the manufacture of pH-sensitive microelectrodes using a liquid proton carrier. It is believed that this method has advantages over other published methods since it allows direct visualization and control of major steps during electrode fabrication and overcomes several problems often encountered using more conventional methods. Microelectrodes fabricated using this technique have been used to measure the in situ pH of venom from two myrmicine ants, Tetramorium caespitum (L.) and Myrmica ruginodis (N.).

Animals↗

PubMed1988

Extra- and intracellular hydrogen ion-selective microelectrode based on neutral carriers with extended pH response range in acid media.

A series of new neutral hydrogen ion carriers suitable for application in H+-selective microelectrodes is presented. One carrier (ETH 1907) proves to be superior to tridodecylamine currently very much in use. Microelectrodes based on ETH 1907 in an optimized membrane composition exhibit a linear dynamic response function from pH 2 to 9 extended into the acidic range, a response time less than or equal to 5 s, and a resistance of about 35 G omega for a tip diameter of about 1 micron. This makes the electrode suitable for measurements at normal physiological intracellular pH as well as in acid physiological media. Measurements using this microelectrode in proximal tubule cells of isolated perfused frog kidney are presented.

Animals↗

PubMed1985

Construction of K+- and Na+-sensitive theta-microelectrodes with fine tips: an easy method with high yield.

A new method is described to prepare theta-microelectrodes with tips up to 0.15 micron diameter controlled under scanning electron microscope. K+- and Na+-sensitive resins were tested. Method features are the following: i) hard drying of the glass, ii) rehydration of one channel and weak wetting of the other with a three-methylchlorosilane solution before pulling, iii) simultaneous presence of water and silane in the two channels during pulling, iv) gradual silanization from the tip to the shank. Selective and conventional channels did not affect each other and no displacements of resins were observed. The change of potential difference of the selective channel was more than -50 mV/decade. Apical membrane potentials and cell Na+ and K+ activities of the epithelial cells of rabbit gall-bladder (cell diameter: 5-10 micron) were measured with these theta-microelectrodes and with single-barrel microelectrodes of similar tip size: results obtained were not significantly different.

Animals↗

PubMed1979

Active and passive properties of rabbit descending colon: a microelectrode and nystatin study.

The electrical properties of the basolateral membrane of rabbit descending colon were studied with microelectrode methods in conjunction with the polyene antibiotic nystatin. Two problems were examined: (i) the relative distribution of tight junctional, apical membrane and basolateral membrane resistances, and (ii) the ionic basis of the basolateral membrane potential. Intracellular K+ activity (K+) was measured using liquid ion exchanger microelectrodes ((K+) = 76 +/- 2 MM) and was found not to be in equilibrium with the basolateral membrane potential. In order to measure membrane resistances and to estimate the selective permeability of the basolateral membrane, the apical membrane was treated with nystatin and bathed with a K2SO4 Ringer's solution which was designed to mimic intracellular K+ composition. This procedure virtually eliminated the resistance and electromotive force of the apical membrane. Shunt resistance was calculated by two independent methods based on microelectrode and transepithelial measurements. Both methods produced similar results (Rs = 691 +/- 63 omega cm2 and 770 +/- 247 omega cm2, respectively). These findings indicate that the shunt has no significant selectivity, contrary to previous reports. Native apical membrane resistance was estimated as 705 +/- 123 V cm2 and basolateral membrane resistance was 95 +/- 14 V cm2. To estimate basolateral membrane selectivity, the serosa was bathed in a NaCl Ringer's solution followed by a series of changes in which all or part of the Na+ was replaced by equimolar amounts of K+. From measures of bi-ionic potentials and conductance during these replacements, we calculated potassium permeability and selectivity ratios for the nystatin-treated colon by fitting these results to the constant field equations. By correcting for shunt conductance, it was then possible to estimate the selective permeability of the basolateral membrane alone. Selectivity estimates were as follows: PNa/PK = .08 and PCl/ PK = .07 (uncorrected for shunt) and PNa/PK = .04 and PCl/PK = .06 (basolateral membrane alone). In a second set of experiments, evidence for an electrogenic Na+ pump in the basolateral membrane is presented. A small ouabain-sensitive potential could be generated in the nystatin-treated colon in the absence of chemical or electrical gradients by mucosal, but not serosal, addition of NaCl. We conclude that this electrogenic pump may contribute to the basolateral membrane potential; however, the primary source of this potential is "passive": specifically, a potassium gradient which is maintained by an "active" transport process. An appendix compares the results of nystatin experiments to amiloride experiments which were conducted separately on the same tissues. The purpose of this comparison was to develop a comprehensive model of colonic transport. The analysis reveals a leak conductance in the apical membrane and the presence of an amiloride-insensitive conductance pathway.

Animals↗

PubMed1978

Biological and artificial ion exchangers: electrical measurements with glass microelectrodes.

Biological (stratum corneum) and artificial (cation-exchange resin beads, Bio-Rad AG 50W-X2) ion exchangers were impaled by glass microelectrodes filled with KCl solution. The electrical potential difference recorded in these structures in reference to the external bathing medium was shown to be dependent on the KCl concentration of both the external and the microelectrode filling solutions. The potentials were interpreted on the grounds of the fixed charge theory of membrane potentials as a consequence of two phase boundary potentials (Donnan potentials), one at the matrix-external solution interface and the other at the matrix-microelectrode solution interface. The contribution of a diffusion component for the recorded potential was considered.

Animals↗

PubMed1983

Penetration of substances into tumor tissue: a methodological study with microelectrodes and cellular spheroids.

A new method was tested for studies of penetration of substances into tumorlike tissue. The penetration of the ions K+, Cl-, and Ca2+ through several layers of tumor cells was demonstrated by using double barrelled, ion sensitive microelectrodes with extra thin tip diameters. Spheroids consisting of human glioma, U-118 MG, and human thyroid cancer, HTh-7, cells were used as models of tumor tissue. A microelectrode was inserted into the center of a spheroid. Thereafter, the concentration of the test substance was increased in the surrounding medium. The change in concentration inside the spheroid was recorded and the penetration pattern evaluated. All three types of tested ions penetrated easily through the spheroids. The K+ ions penetrated most efficiently, and the Ca2+ ions showed the slowest penetration. The Ca2+ ions penetrated somewhat more slowly in the U-118 MG spheroids (which had rather small extracellular spaces) than in the HTh-7 spheroids (which had larger extracellular spaces). Ion sensitive electrodes, which are easily available, were used in this study only to demonstrate the principle. We hope that the method described can be used for penetration studies of various substances. For example, all substances that can be detected by enzyme microelectrodes could be studied. The main advantage of the method is that the complete penetration pattern can be studied as a function of time in individual spheroids. Previously described methods require histological procedures for each analyzed penetration time.

Biological Transport↗

PubMed2004

In vivo intravascular electric impedance spectroscopy using a new catheter with integrated microelectrodes.

Interventional techniques are necessary, which allow the characterization of intravascular pathological processes. Electric impedance spectroscopy (EIS) can provide cellular information of biological tissue. We tested the feasibility of intravascular EIS by using a new impedance catheter system with integrated microelectrodes in an experimental animal model. Eighteen stents were implanted into the iliac arteries of female New Zealand White rabbits (n = 11) to induce intimal proliferation. After 14, 28 and 56 days the electric impedance was measured inside and outside of the stented arterial segments by using a balloon catheter with four integrated microelectrodes. The impedance was recorded at a frequency ranging from 1 Hz to 1 MHz. After the measurements, the stents were explanted and histomorphometry was performed. The impedance inside and outside the stent was analysed and compared with the histomorphometric data. Fourteen (n = 6), 28 (n = 5) and 56 (n = 6) days after stent implantation the difference of the electrical impedance between the native and the stented iliac artery segment increased from -924 +/- 715 Ohm to 3689 +/- 1385 Ohm (14 days vs. 28 days; p < 0.05) and 8637 +/- 2881 Ohm (14 days vs. 56 days; p < 0.05), respectively. The increase of the electrical impedance corresponded to an increased neointimal proliferation in the stented arterial segment of 3.6% +/-0.7% after 14 days, 8.4% +/- 4.8% after 28 days (14 days vs. 28 days; p < 0.05) and 10.0% +/- 4.1% after 56 days (14 days vs. 56 days; p < 0.01). Intravascular EIS can be performed by a balloon catheter with integrated microelectrodes and allows the detection of neointimal proliferation after stent implantation.

Animals↗

PubMed1987

Measurement of intracellular chloride activity in mouse liver slices with microelectrodes.

Steady-state membrane potential (Vm) and intracellular Cl- activity (aCli) were measured with double-barreled Cl(-)-selective microelectrodes in mouse liver slices. In bathing solutions (33.8 degrees C) containing pyruvate, glutamate, fumarate, and glucose, Vm and aCli were -27.6 +/- 1.0 mV and 32.6 +/- 1.5 mM, respectively. This apparent value of aCli exceeded the level required for passive distribution of this ion (aCleq = 26.4 +/- 1.3 mM) by 6.2 +/- 1.0 mM. This difference was essentially unchanged in experiments where (i) Na+ was replaced by choline, (ii) HCO3- was removed, and (iii) Cl- was replaced by gluconate. These data argue against the presence of Na+- or HCO3(-)-coupled Cl- transport mechanisms in the plasma membrane of mouse liver cells. This implies that aCli is in fact at equilibrium and interference with the response of Cl(-)-selective microelectrodes by intracellular anions is responsible for the apparent difference between aCli and aCleq. We found that Cl(-)-selective microelectrodes containing Corning 477315 ligand are sensitive to taurocholate, a representative bile salt. Their selectivity to taurocholate is about 60-times their selectivity towards Cl-. This suggests that interference of bile acids at concentrations normally present in hepatocytes with determinations of aCli can account for the apparent difference aCli-aCleq.

Animals↗

PubMed1994

Intracellular microelectrode membrane potential measurements in tobacco cell-suspension protoplasts and barley aleurone protoplasts: interpretation and artifacts.

Intracellular microelectrode measurements in plant cell protoplasts have been widely used to study hormone signal transduction processes. However, the interpretation and reliability of such measurements are largely dependent on a detailed evaluation of the measurement conditions, as investigated in the present paper. Upon microelectrode penetration of tobacco cell suspension protoplasts and of barley aleurone protoplasts a fast negative going impalement-induced potential transient of less than a few ms duration could be observed. After reaching a steady-state potential at the ms time scale the measured potential hyperpolarized again and, in most cases, subsequently depolarized to a new steady-state value. Analysis of the electrical equivalent circuit of the measurement configuration showed that the occurrence of the impalement-induced potential transient indicates that these measurements suffer from a microelectrode-induced shunt resistance which loads the measurement. In addition, it is shown that the peak-value of the potential transient is the most reliable indicator of the true membrane potential and of true membrane potential changes of the protoplast, since this value is rather membrane resistance independent. For correct interpretation of steady-state measurements of membrane potential and stimulus-induced membrane potential changes data on membrane and shunt resistance are essential. As an example of the measurement of membrane potential changes the effects of 1-NAA on measured potential values in tobacco protoplasts and the effect of extracellular pH changes on barley aleurone protoplasts are analyzed with regard to the above described conclusions.

Hordeum↗

PubMed1985

Ca2+-selective microelectrodes.

Ca2+-selective microelectrodes based on the synthetic neutral carrier ETH 1001 can be used for quantitative intracellular measurements of resting Ca2+-activities and of slowly changing Ca2+-levels (response time in the order of seconds). Microelectrodes with tip diameters greater than 0.3 micron show selectivities that yield a detection limit between 10(-8) and 10(-7) M Ca2+ in an intracellular background. The Ca2+-activity is obtained together with electrical parameters of the cell (e.g. cell membrane potential and membrane resistance or conductivity). Simultaneous monitoring of other ion-activities is accessible (double- or multi-barrelled microelectrodes). The Ca2+-determination is extremely local, i.e. it probably does not indicate an averaged cytosolic activity in every situation (e.g. localized transients).

Calcium↗

PubMed1986

Procedures for manufacturing double-barrelled ion-sensitive microelectrodes employing liquid sensors.

Liquid ion-sensitive/selective sensors are available for most inorganic ions of physiological and biochemical importance. In order to measure intracellular ionic activities in relatively small cells, it is advisable to manufacture and use double-barrelled microelectrodes. Procedures for making two types of double-barrelled ion-sensitive microelectrode are described in detail. Such microelectrodes have been used successfully to measure intracellular K+, Cl- and Na+ activities in retinal horizontal cells of fish and body-wall muscles of insect larvae.

Animals↗

PubMed1980

An almost completely shielded microelectrode.

We present a new method of shielding microelectrodes to within 20 micron of the tip. Stray capacity is reduced to less than 50 fF. Ordinary microelectrodes are covered with silver in a vacuum evaporator. Silver is removed from the tip by contact with a ball of mercury. The microelectrode is then insulated with a glass barrel which is sealed by dipping the tip in diluted polystyrene in amyl acetate, or by dipping the electrode in melted wax. The latter method is quick, easy and reliable.

Animals↗

PubMed1989

A simple device for the reliable production of varnish-insulated, high-impedance tungsten microelectrodes.

The construction and operation of a simple device that produces varnish-insulated, high-impedance tungsten microelectrodes for single-unit recording is described. In essence, the device operates as a high-voltage pulse generator whose output creates an arc between a fully insulated tungsten microelectrode and a polished counter-electrode. As a result, insulation is removed reliably and symmetrically from the microelectrode tip. The device is constructed with a minimal outlay of time, skill, and money.

Electrophysiology↗

PubMed1990

Ion-selective microelectrodes suitable for recording rapid changes in extracellular ion concentration.

A method for fabricating double-barrel, ion-selective microelectrodes with fine tips (0.5-1.5 microns) and rapid response times is described. When made into K(+)-selective microelectrodes, the electrodes respond to changes in [K+]o with a time constant of 70-95 ms. The electrical response of these electrodes to common-mode voltages can be made to have a time constant of less than 2 ms, which minimizes electrical artifacts from field potentials. The application of these microelectrodes to the measurement of rapid, transient changes in retinal [K+]o is presented.

Animals↗