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PubMed1985

Comparative measurements of potassium and chloride with ion-sensitive microelectrodes and x-ray microanalysis in cultured skeletal muscle fibers.

Data of the intracellular electrolyte concentration of potassium and chloride in cultured muscle cells measured by x-ray analysis were compared by using the different activity coefficients with intracellular potassium and chloride activities measured with double-barrelled microelectrodes. By using an activity coefficient of 0.6, 95% of the potassium microelectrode measurements are in accordance with the x-ray analysis values, in spite of a scattering of the values. Membrane potential and intracellular potassium values are linearly related. x-ray analysis and ion-sensitive microelectrodes measured the cytoplasmic chloride in the same range. Taking into account known activity coefficients, an error of 25% must be assumed with the intracellular chloride measurements. However, x-ray analysis and ion-sensitive microelectrode investigations are reliable tools to study intracellular potassium and chloride changes, which play an important role in membrane characteristics.

Animals↗

PubMed1990

Biphasic voltage relaxation pattern observed in cells of Eremosphaera viridis after injection of charge-pulses of short duration: detection of tip clogging of intracellular microelectrodes by charge-pulse technique.

Charge pulse experiments performed on the peat-bog alga Eremosphaera viridis revealed an unusual voltage relaxation behaviour. Injection of charge pulses of 1 microseconds duration resulted in an immediate charging of the membranes (time constant of the order of 40 ns). Nevertheless, the potential-measuring microelectrode recorded an exponential increase in membrane voltage with a time constant of about 1.3 ms. The maximum voltage value was recorded after about 3 ms, followed by an exponential decay with a time constant of about 9.6 ms. This biphasic time course was independent of the amplitude of the injected charge and of the location of the impaled microelectrodes in the vacuole. Centrifuged cells in which the chloroplasts and the other organelles were pelleted in one part of the cells showed the same electrical response. Electrical breakdown of the cell membranes resulted in the disappearance of the biphasic voltage response. In this case only the decaying relaxation process could be recorded with a time constant of 3 ms. After resealing of the membranes the original biphasic relaxation response was restored. Increasing concentrations of KCl in the bathing medium reduced both time constants almost correspondingly. The experimental findings were evaluated with an electrical equivalent circuit. Theoretical analysis with reference to the experimental data suggested that the delayed voltage response of the potential-recording electrode resulted from a membrane seal across the tip of this electrode. The resistance of this seal was calculated to be about 400 M omega. The specific resistances and capacitances of tonoplast and plasmalemma membranes were calculated from the decaying part of the biphasic relaxation curves. The average values were found to be 2.58 omega.m2 and 5 mF.m-2. The investigations reported here suggest that charge pulse experiments can be generally used for the detection of membrane and cytoplasmic material clogging of the tip of intracellular microelectrodes, a problem with which most electrophysiologists are faced when interpreting data obtained from impaled microelectrodes.

Cell Membrane↗

PubMed1986

Neuronal activity evoked by chronically implanted intracortical microelectrodes.

The averaged evoked compound action potentials (AECAPs) were recorded from the ipsilateral pyramidal tract of awake, unrestrained cats before, during, and after continuous electrical stimulation of the cerebral cortex via chronically implanted activated iridium or platinum-30% iridium (Pt30%Ir) microelectrodes. After stimulating 24 h at 20 pulses per second (pps), using charge-balanced, 200-microseconds pulse pairs of 40 to 80 microA (400 to 800 microC/cm2, 8 to 16 nC/phase (ph), 2 to 4 A/cm2), there was a transient elevation of the threshold of the early (direct) and of the alte (transynaptic) components of the AECAP. After cessation of continuous stimulation at 80 microA, the threshold of the early component of the AECAP remained elevated for as long as 24 h and the late component as long as 4 days, indicating significant but reversible depression of the electrical excitability of cortical neurons close to the microelectrodes. In three cats stimulated 23 h/day for 1 week, the AECAP also recovered to their prestimulus threshold. In contrast, pulsing for 24 h at 320 microA (3200 microC/cm2, 64 nC/ph, 16 A/cm2) produced marked elevation of the threshold of the AECAPs which was not reversed by 7 to 12 days after termination of intracortical stimulation. The electrical excitability of neurons adjacent to (unpulsed) microelectrodes 2 mm from the pulsed electrode was not affected. The observations reported here, in conjunction with the histologic results reported in the companion paper, indicate that both the Pt30%Ir and the iridium microelectrodes can be operated safely at currents to at least 80 microA, charge/ph of 16 A/cm2, and a charge density of 800 microC/cm2 X ph. However, on the basis of the electrophysiologic criteria, both types appear to be unsafe when pulsed at 320 microA (64 nC/ph, 3200 microC/cm2 X ph, 16 A/cm2).

Action Potentials↗

PubMed1994

A new method for manufacturing carbon-fibre microelectrodes.

A new, fast and low-cost method using a carbon-fibre microelectrode is proposed. The microelectrode is constructed by glueing one or more carbon fibres (5 mm in diameter) with a silver resin on a silver wire or stainless steel tube. The characteristics of that microelectrode have been measured and compared with those of commercially available glass carbon-fibre microelectrodes. According to our measures, the impedance, capacity and current noise were lowered which permits these electrodes to be used in voltammetry and in detection of very low currents generated during formation of antigen-antibody complex.

Carbon↗

PubMed1989

An ion-selective microelectrode for bile salts.

The development of a prototype ion-selective microelectrode for bile salts is described. The microelectrodes demonstrated Nernstian response (59 +/- 10 mV dec-1) with sodium deoxycholate in Tris (0.2 M, pH 9.0, 15-30 degrees C), HEPES (0.13 M, pH 7.5), and bicarbonate buffers (0.1 M, pH 7.5) (25 +/- 0.1 degrees C) were stable for several days, and the responses were highly reproducible. The microelectrodes were selective for bile salts over the physiologically important inorganic anions, bicarbonate and chloride. The response to sodium cholate (42 mV dec-1) was consistently lower than the ideal response (59 mV dec-1). This ion-selective microelectrode may show promise as a useful tool for the determination of intracellular bile salt activity.

Bile Acids and Salts↗

PubMed1980

Resistive artifacts in liquid-ion exchanger microelectrode estimates of Na+ activity in epithelial cells.

In experiments on the rabbit urinary bladder epithelium we have identified an electrical artifact in certain liquid ion-sensitive microelectrodes. This artifact arises from the high electrical resistance of the ion-sensitive resins which in some cases are comparable to the resistance of the microelectrode glass wall. For Na+-sensitive microelectrodes this situation led to shunting of the exchanger potential and consequently artifactually high calculations of intracellular Na+ in the rabbit urinary bladder epithelium. A method for minimizing this shunting effect is described. After reduction of the shunt the frequency response of the Na+-sensitive microelectrode was increased and the estimated ai Na+ was decreased to 7 mM.

Animals↗

PubMed1996

Simultaneous measurements of intracellular pH in the leech giant glial cell using 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and ion-sensitive microelectrodes.

We have employed two independent techniques to measure the intracellular pH (pHi) in giant glial cells of the leech Hirudo medicinalis, using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and double-barreled neutral-carrier, pH-sensitive microelectrodes, which also record the membrane potential. We have compared two procedures for calibrating the ratio of the BCECF signal, excited at 440 nm and 495 nm: 1) the cell membrane was H(+)-permeabilized with nigericin in high-K+ saline at different external pH (pHo) values, and 2) the pHi of intact cells was perturbed in CO2/HCO3(-) -buffered saline of different pH, and the BCECF ratio was calibrated according to a simultaneous microelectrode pH reading. As indicated by the microelectrode measurements, the pHi did not fully equilibrate to the pHo values in nigericin-containing, high-K+ saline, but deviated by -0.12 +/- 0.02 (mean +/- SEM, n = 37) pH units. In intact cells, the microelectrode readings yielded up to 0.15 pH unit lower values than the calibrated BCECF signal. In addition, larger dye injections into the cells (> 100 microM) caused an irreversible membrane potential loss indicative of some damage to the cells. The amplitude and kinetics of slow pHi changes were equally followed by both sensors, and the dye ratio recorded slightly higher amplitudes during faster pHi shifts as induced by the addition and removal of NH4+.

Animals↗

PubMed1998

In vivo microelectrode track reconstruction using magnetic resonance imaging.

To obtain more precise anatomical information about cortical sites of microelectrode recording and microstimulation experiments in alert animals, we have developed a non-invasive, magnetic resonance imaging (MRI) technique for reconstructing microelectrode tracks. We made microelectrode penetrations in the brains of anesthetized rats and marked sites along them by depositing metal, presumably iron, with anodic monophasic or biphasic current from the tip of a stainless steel microelectrode. The metal deposits were clearly visible in the living animal as approximately 200 microm wide hypointense punctate marks using gradient echo sequences in a 4.7T MRI scanner. We confirmed the MRI findings by comparing them directly to the postmortem histology in which the iron in the deposits could be rendered visible with a Prussian blue reaction. MRI-visible marks could be created using currents as low as 1 microA (anodic) for 5 s, and they remained stable in the brains of living rats for up to nine months. We were able to make marks using either direct current or biphasic current pulses. Biphasic pulses caused less tissue damage and were similar to those used by many laboratories for functional microstimulation studies in the brains of alert monkeys.

Animals↗

PubMed1990

Copper determination in urine by flow injection analysis with electrochemical detection at platinum disk microelectrodes of various radii.

The incorporation of platinum disk microelectrodes of various radii (2.5-50 microns) in a well-jet flow cell offers reduced limits of detection for the determination of copper in urine by flow injection analysis compared with standard methods based on a conventional sized glassy carbon disk macroelectrode (radius 1.5 mm), in a thin-layer cell. The radius of the platinum disk microelectrode was found to be critical with respect to both the limit of detection and flow rate dependence. An optimal radius value of 28 microns was found with detection limits increasing with both larger and smaller electrode radii. In contrast, as theoretically expected, a diminished flow rate dependence was observed the smaller the radii of the platinum disk microelectrodes. Sample cleanup and preparation is conveniently achieved by the use of Sep-Pak cartridges and formation of a copper dithiocarbamate complex. The metal complex is easily oxidized at platinum disk microelectrodes in acetonitrile, which was the solvent used in the flow injection method of analysis.

Copper↗

PubMed2000

Ceramic-based multisite microelectrodes for electrochemical recordings.

This paper describes the development and characterization of ceramic-based multisite arrays for electrochemical recordings in biological systems. These electrodes represent a parallel technology to the design of microelectrodes using silicon substrates. The ceramic substrates are stronger than silicon and are nonconducting, which makes them better suited for in vivo electrochemical measurements. The current designs are based on formation of four-site (50 x 50 microns with 200 microns spacing) electrodes on ceramic wafers using photolithography. The recording sites and connecting lines are made of Pt with a polyimide coating to insulate the connecting lines. The resulting electrodes are cut from the wafers producing a 1 cm length microelectrode that tapers to a approximately 2-5 microns tip. Electrochemical measures of dopamine and hydrogen peroxide support that the sensitivity, selectivity, and response characteristics of the electrodes exceed those of previously published silicon substrate-based microelectrodes. This is the first demonstration of microarrays formed from ceramic substrates, and the data presented support the hypothesis that these microelectrodes may be useful for a variety of neurochemical and electrophysiological applications. Preliminary in vivo electrochemical recordings are presented.

Animals↗

PubMed1985

Cytosolic free calcium-ion concentration in cleaving embryonic cells of Oryzias latipes measured with calcium-selective microelectrodes.

Calcium-selective microelectrodes were used to measure the free calcium-ion concentration ([Ca2+]i) in early-cleaving embryonic cells of the golden medaka, Oryzias latipes, a fresh water teleost fish. Embryos could be dechorionated as early as the four-cell stage using a three-step technique consisting of removal of some yolk to enlarge the perivitelline space, partial digestion of the chorion with pancreatin, and removal of the weakened chorion with forceps. Dechorionated embryos underwent cleavage at a normal rate. Intracellular cytosolic [Ca2+]i was monitored by impaling blastomeres first with a microelectrode filled with 5 M potassium acetate to measure membrane potential, and a few minutes later with a calcium-selective microelectrode. During nine rounds of cytokinesis from a total of six different embryos, cytosolic [Ca2+]i remained constant (with apparently random fluctuations of less than +/- 0.1 microM). During two successive cleavages in one embryo, however, [Ca2+]i rose transiently fourfold above the original resting level to 1.32 and 1.20 microM in synchrony with each period of cytokinesis and returned after each rise to submicromolar levels. Because a calcium-selective microelectrode can detect [Ca2+]i changes only in the immediate vicinity of its 2-microns tip, we interpreted these data to suggest that, although [Ca2+]i in most areas of the cytosol remains between 0.01 and 0.40 microM (mean of 0.14 microM), there may be small regions of the cell in which [Ca2+]i undergoes a substantial increase at the time of cleavage. Evidence also is presented to suggest that the membrane potential in these blastomeres undergoes a slow net hyperpolarization during early cleavage stages.

Animals↗

PubMed1983

Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry.

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388D1 and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (E(s)) frequently reported. Upon microelectrode entry into the cell (P388D1), the recorded potential drops to a peak value (E(p)) (mean -37 mV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (E(n)) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (E(s)) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (E(h)) is -30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U.V., A.M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288 for other change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of E(p), E(n), E(h), E(s), and membrane resistance (R(m)) measured for the other macrophages were similar to those of P388D1. From these results we conclude that E(p) is a better estimate of the true membrane potential of macrophages than E(s), and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes.

Animals↗

PubMed1996

Histopathologic and physiologic effects of chronic implantation of microelectrodes in sacral spinal cord of the cat.

Active microelectrodes were implanted for a period of 2 weeks to 3 months into the sacral spinal cord of 10 male cats in order to test the feasibility and the safety of discrete stimulation of the parasympathetic preganglionic nucleus for future clinical applications of microelectrode technology in micturition control. An array of four 50 microns-diameter iridium microelectrodes was inserted beneath the dura in each cat. At weekly intervals, bladder pressure was measured as hydrostatic pressure on an intraluminal catheter. At the end of the period, histopathology was evaluated with serial transverse epoxy sections. Observations included diffuse and focal axonal degeneration in white matter and possible neuronal loss around the electrode in the gray matter, meningeal ensheathment of the shafts, and occasional aseptic inflammation of tissue and apparent movement of the electrodes after implantation. Increased bladder pressure responses to individually pulsed electrodes located within the sacral parasympathetic nucleus were not consistent, and, surprisingly, at least 2 different sites were also effective. As long as 3 months after implantation, in 2 out of 5 animals, pulsing of electrodes consistently produced micturition. We conclude that while microelectrode implants are feasible, further modifications in electrode design are needed to eliminate movement and inflammation.

Animals↗

PubMed1992

Regeneration microelectrode array for peripheral nerve recording and stimulation.

A microelectrode array capable of recording from and stimulating peripheral nerves at prolonged intervals after surgical implantation has been demonstrated. The microelectrode array, fabricated on a silicon substrate perforated by multiple holes (referred to as via holes), is implanted between the ends of a surgically severed nerve. Regenerating tissue fixes the device in place to provide a stable mapping between the microelectrodes and the axons in the nerve. Processes were developed for the fabrication of thin-film iridium microelectrodes, micromachined via holes, and silicon nitride passivation layers. All fabrication methods were designed to be compatible with standard CMOS/BiCMOS processes to allow for on-chip signal processing circuits in future designs. Such arrays, implanted in the peroneal nerves of rats, were used to record from and stimulate the nerves at up to 13 months postoperatively.

Animals↗

PubMed1997

A characterization of the effects on neuronal excitability due to prolonged microstimulation with chronically implanted microelectrodes.

Localized, long-lasting stimulation-induced depression of neuronal excitability (SIDNE) is a consequence of prolonged, high-frequency microstimulation in the central nervous system (CNS). It represents a persisting refractory state in the neurons and axons near the stimulating microelectrode, that occurs in the absence of histologically detectable tissue injury. It does not involve a change in synaptic efficacy and, in this respect, it differs from the more familiar phenomenon of long-term depression (LTD). Although SIDNE is ultimately reversible (after several days), it must be taken into account in the design of neural prostheses based on microstimulation in the central nervous system and in animal studies that require prolonged microstimulation in the CNS. In this study, we have characterized the phenomenon, using as the paradigm, iridium microelectrodes implanted chronically in the cat's posteroventral cochlear nucleus. Although the SIDNE may persist for several days after the end of the stimulation protocol, it does not become more severe from day to day when the stimulation protocol is repeated on successive days. The severity of the SIDNE is strongly dependent upon both the instantaneous frequency and the duty cycle of the electrical stimulation. The character of the SIDNE, including its localization to the immediate vicinity of the stimulating microelectrodes, suggests that the phenomenon is a direct consequence of the prolonged electrical excitation of the neurons close to the microelectrode. The problem of designing microstimulation systems that allow high-frequency stimulation of a neural substrate, while minimizing SIDNE are discussed.

Animals↗

PubMed1983

A new design of double-barrelled microelectrodes for intracellular pH-measurement in vivo.

Intracellular pH (pHi) is one important regulator of cellular function. Most determinations of pHi in vivo have been performed by using indirect methods, e.g. CO2/HCO3 or DMO techniques, since no suitable direct method for reliable in vivo measurements have been available. In the present study a new type of double-barrelled microelectrode for simultaneous in vivo measurements of pHi and membrane potential (Em) is presented. The electrode was constructed on the basis of a combined recessed- and pencil-tip design. The tip diameter of the double-barrelled microelectrode was about 1.5 microns. The response of the pH channel was 55-60 mV/pH unit and the response time was between 30 s and 1 min. In vivo measurements of pHi and Em of rabbit skeletal muscle fibers are presented. A mean pHi of 7.00 (in 8 animals) at a membrane potential of -90.3 mV (arterial pH: 7.30, arterial PCO2: 6.39 kPa) was obtained. The new design of pH microelectrode offers some advantages over previously described microelectrodes and is well suited for in vivo measurements.

Animals↗

PubMed1987

Cell contamination due to the use of carrier-based microelectrodes.

When using microelectrodes for intracellular ion activity studies, some uncertainties such as interference from cytosolic components at the microelectrode, cell damage, and cell contamination may arise. A model, which treats kinetic processes of the loss of carriers from the membrane phase of microelectrodes into the cytosol and cell membrane, is used for an estimation of the extent and time course of contamination by impaled ion-selective microelectrodes. An isolated model cell consisting of a plasma membrane surrounding a cytosolic milieu is assumed. The results of its considerations represent a worst case situation, in which significant contamination of the cell membrane of such a small isolated single cell might occur during time periods of electrophysiological experiments. In more complex situations, such as in intact tissues, the equilibrium membrane concentrations may be substantially less.

Animals↗

PubMed1986

Measurements of intracellular pH in Necturus antral mucosa by microelectrode technique.

Intracellular pH (pHi) was measured in the surface epithelial cells of Necturus antrum using pH-sensitive intracellular microelectrodes. Electrodes were prepared by filling 10- to 20-M omega resistance glass microelectrodes with a H+ ion-selective exchange resin and calibrated before use in solutions of known pH 4.0-8.0. The electrode response (n = 15) was linear (r = 0.93; P less than 0.001) with a slope of 52.1 +/- 2.3 mV/pH unit. Antral mucosa was mounted in a modified Ussing chamber and pHi was determined from the difference between the potentials recorded by intracellular H+-selective and conventional microelectrodes. These measurements of pHi were validated by examining the response of the intracellular microelectrodes to 1) depolarization of the cell membrane produced by K+ substitution for Na+, and 2) alkalinization and acidification of pHi produced by NH+4 substitution for Na+ in the bathing solutions. In tissues bathed with HCO-3-Ringer solution (pH 7.0), the mean pHi was 7.34 +/- 0.02 with a range from 7.24 to 7.43. In N-2-hydroxymethylpiperazine-N' -ethanesulfonic acid (HEPES)-Ringer solution (pH 7.0), pHi was reduced to 7.02 +/- 0.05 (P less than 0.01). Acidification of the luminal solution to pH 6.8 with CO2 produced a 0.22 +/- 0.04-pH unit fall in pHi (P less than 0.001). In contrast, acidification to pH 4.0 with HCl had no significant effects on pHi. These findings indicate that HCO-3 may play an important role in pHi regulation in this tissue. In addition, they suggest that, in contrast to CO2, the surface epithelial cells of Necturus antrum are relatively resistant to acidification by luminal HCl.

Animals↗