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A technique for brain temperature control during ischemia, suitable for measurements with ion-sensitive microelectrodes.

A technique is described for maintaining rat brain temperature constant during ischemia, a technique that also allows measurements with, and calibration of, ion-sensitive microelectrodes under defined temperature conditions. The brain temperature is controlled by a stream of air of defined temperature and humidity, which is perfused through a box enclosing the animal's head. A device for calibration of ion-sensitive microelectrodes is temperature controlled by similar principles. The air stream is delivered by a heater/humidifier that is standard in many commercial respirators/ventilators. When the relative humidity of the air stream is greater than 98%, the neocortical temperature can be maintained within less than 0.5 degrees C during 15 min of ischemia. The biological applicability of the technique is discussed.

Animals↗

Microelectrodes in medicine.

The chief applications of microelectrodes in medicine are in experimental studies of tissue and cell behaviour and of metabolism. Microprobes have been developed for a wide range of specific substrates and for the detection of bioelectric potentials. They have provided information that now forms the basis of much of our understanding of biological phenomena. Their inherent characteristics, such as fragility and 'drift', render them unsuitable for general clinical application except at the surface of organs. The heterogeneity of tissues makes it necessary to specify the exact anatomical location of microelectrode measurements and the probes themselves must be of a form that neither distorts, damages, nor stimulates reactions from, the cells or blood vessels with which they are in contact.

Animals↗

Stability of the interface between neural tissue and chronically implanted intracortical microelectrodes.

The stability of the interface between neural tissue and chronically implanted microelectrodes is very important for obtaining reliable control signals for neuroprosthetic devices. Stability is also crucial for chronic microstimulation of the cerebral cortex. However, changes of the electrode-tissue interface can be caused by a variety of mechanisms. In the present study, intracortical microelectrode arrays were implanted into the pericruciate gyrus of cats and neural activities were recorded on a regular basis for several months. An algorithm based on cluster analysis and interspike interval analysis was developed to sort the extracellular action potentials into single units. We tracked these units based on their waveform and their response to somatic stimulation or stereotypical movements by the cats. Our results indicate that, after implantation, the electrode-tissue interface may change from day-to-day over the first 1-2 weeks, week-to-week for 1-2 months, and become quite stable thereafter. A stability index is proposed to quantify the stability of the electrode-tissue interface. The reasons for the pattern of changes are discussed.

Action Potentials↗

Endoneural selective stimulating using wire-microelectrode arrays.

In acute experiments eight 5- to 24-wire-microelectrode arrays were inserted into the common peroneal nerve of the rat, to investigate whether the electrodes could selectively stimulate motor units of the extensor digitorum longus (EDL) muscle. Twitch-force-recruitment curves were measured from the EDL for each array electrode. The curves were plotted on a double-logarithmic scale and parameterized by the low-force slope (which represents the power p in the power-law relationship of force F versus stimulus current I, or F approximately I(p)) and the threshold current. The slopes and threshold currents measured with array electrodes did not differ significantly from those obtained with randomly inserted single wire-microelectrodes. This indicates that, although involving a more invasive insertion procedure, electrode arrays provide neural contacts with low-force recruitment properties similar to those of single wires. Array results revealed partial blocking of neural conduction, similar to that reported with microneurographic insertion with single needles. The efficiency of the array was defined as the fraction of array electrodes selectively contacting a motor unit and evoking the corresponding threshold force. Efficiency thus expresses the practical value of the used electrode array in terms of the total number of distinct threshold forces that can be stimulated by selecting the appropriate electrodes. The eight arrays were capable of evoking threshold forces selectively with an average efficiency of 0.81 (or 81%).

Animals↗

Microelectrode array fabrication by electrical discharge machining and chemical etching.

Wire electrical discharge machining (EDM), with a complementary chemical etching process, is explored and assessed as a method for developing microelectrode array assemblies for intracortically recording brain activity. Assembly processes based on these methods are highlighted, and results showing neural activity successfully recorded from the brain of a mouse using an EDM-based device are presented. Several structures relevant to the fabrication of microelectrode arrays are also offered in order to demonstrate the capabilities of EDM.

Action Potentials↗

Extracellular recordings from patterned neuronal networks using planar microelectrode arrays.

Neuronal cell networks have been reconstructed on planar microelectrode arrays (MEAs) from dissociated hippocampal pyramidal neurons. Microcontact printing (microCP) and a photoresist-liftoff method were used to selectively localize poly-L-lysine (PLL) on the surface of MEAs. Haptotaxis led to the organization of the neurons into networks localized adjacent to microelectrodes. Various grids of PLL with 2-25-microm-wide lines spaced by 50-200 microm with 15-25-microm nodes at intersection points were used to guide cell body attachment and neurite outgrowth. Bursting activity with spike amplitude attenuation was observed, and multichannel recordings detected instances of coincident firing activity. Finally, we present here an extracellular recording from a approximately 2 microm bundle of guided neurites.

Action Potentials↗

Alkaline secretion by frog gastric glands measured with pH microelectrodes in the gland lumen.

1. In the present work we have measured the pH of the secreted fluid within the gland lumen of isolated but intact gastric mucosa of Rana esculenta. Tissues were mounted in a double chamber allowing continuous perfusion of the mucosal and serosal compartment, and the measurements were made with double-barrelled pH glass microelectrodes inserted into the glands from the serosal surface under microscopic inspection. 2. During inhibition of H+ secretion by cimetidine (100 microM) the luminal gland pH (pHgl) averaged 7.60 +/- 0.05 pH units (mean +/- s.e.m.; n = 35), a value significantly higher than bath solution pH (7.45 +/- 0.02; P < 0.001) and also higher than intracellular pH of oxyntopeptic cells (pHi), which averaged 7.53 +/- 0.06 (n = 18). 3. Stimulation of acid secretion with histamine (500 microM) reversibly decreased pHgl to values which could be as low as 2.5. Together with electrophysiological criteria this response was routinely used to verify the proper location of the microelectrode tip within the gland lumen. 4. Stimulation with carbachol (100 microM) or pentagastrin (50 microM) in the presence of cimetidine rapidly and reversibly increased pHgl by 0.10 +/- 0.01 pH units (n = 24; P < 0.001) and 0.09 +/- 0.02 pH units (n = 6; P < 0.05), respectively. 5. The observation that gastric gland fluid is more alkaline than the bath solutions and that carbachol or pentagastrin further alkalinize it strongly suggests that oxyntopeptic cells participate in gastric alkaline secretion at least under cholinergic stimulation.

Animals↗

The regulation of intracellular Mg2+ in guinea-pig heart, studied with Mg(2+)-selective microelectrodes and fluorochromes.

Because of the reported presence of a Na(+)-Mg2+ exchanger in guinea-pig but not in ferret myocardium, the Mg2+ extrusion mechanism in guinea-pig myocardium has been reinvestigated using Mg(2+)- and Na(+)- selective microelectrodes and the fluorochromes mag-fura-2 and -5. The mean [Mg2+]i measured with microelectrodes in trabeculae or papillary muscles was 0.72 mmol/l (n = 22, thirteen experiments; range 0.42-1.23 mmol/l). Increasing [Mg2+]o from 0.5 mmol/l to either 10.5 or 20 mmol/l caused small increases in [Mg2+]i. Decreasing [Na+]o by 50% had no effect on the [Mg2+]i and there was no change in [Na+]i on increasing [Mg2+]o from 0.5 to 10.5 mmol/l. Varying pHo or changing pHi with NH4Cl did not influence the [Mg2+]i. In vitro calibration of mag-fura-2 and -5 using the ratio method gave values for K'd (experimentally determined dissociation constant) of 22.2 +/- 2.7 (mean +/- S.D., n = 7) and 25.7 +/- 1.3 (n = 4) mmol/l respectively. Mag-fura-2 reacted to physiological concentrations of Ca2+ and mag-fura-5 to changes in pH. In isolated myocytes, Na+ removal gave an apparent increase of [Mg2+]i with mag-fura-2 but not with mag-fura-5. However, when the pHi was altered with NH4Cl mag-fura-5 showed an apparent decrease in [Mg2+]i on application and an apparent increase on removal, with a time course similar to the pHi changes. It is concluded that Mg2+ extrusion in guinea-pig myocardium is not via a Na(+)-Mg2+ exchanger. The use of mag-fura-2 and -5 are limited in their application because of Ca2+ and H+ sensitivity respectively.

Animals↗

Intracellular Mg2+ regulation in voltage-clamped Helix aspersa neurones measured with mag-fura-2 and Mg(2+)-sensitive microelectrodes.

The extrusion mechanism for intracellular Mg2+ was investigated in voltage-clamped snail neurones using Mg(2+)-sensitive microelectrodes and mag-fura-2. The intracellular free magnesium ion concentration ([Mg2+]1) of snail neurones voltage clamped to -60 mV was estimated to be 0.57 +/- 0.06 mM (mean +/- S.E.M.; n = 12) using Mg(2+)-sensitive microelectrodes and 0.62 +/- 0.05 mM (n = 15) using mag-fura-2. Raising extracellular MgCl2 from 5 to 20 mM caused an average increase in [Mg2+]1 of 0.25 +/- 0.04 mM (n = 7). In three experiments, removing extracellular Mg Cl2 caused an average decrease in [Mg2+]1 of 0.1 mM. Replacing extracellular Na+ with N-methyl-D-glucamine (NMDG) caused a rise in [Mg2+]1 of 1.8 +/- 0.5 mM (n = 7); [Mg2+]1 recovered to resting levels when extracellular Na+ was restored. Iontophoretic injections of MgCl2 were used to raise [Mg2+]1. The rate of recovery from such increases in [Mg2+]1 ¿calculated from the slope of the recovery was inhibited by 85-100% (n = 5) in the absence of extracellular Na2+ compared with control conditions. Raising extracellular Ca2+ from 7 to 35 mM caused a reversible rise in [Mg 2+]1 of 0.4 +/- 0.05 mM (mean +/- S.E.M., n = 7). It was concluded that in snail neurones the main mechanism for [Mg2+]1 extrusion is a Na(+)-Mg2+ exchanger which may be partially inhibited be high extracellular Ca2+ concentrations.

Animals↗

Sodium-selective liquid ion-exchanger microelectrodes for intracellular measurements.

The sodium-selective ligand 1,1,1-tris[1(1)-(2(1)-oxa-4(1)-oxo-5(1)-aza-5(1)-methyl)dodecanyl]propane dissolved in 3-nitro-o-xylene containing a small amount of the lipophilic anion tetrachlorophenyl borate was used as a liquid ion-exchanger in sodium-selective microelectrodes. The microelectrodes gave rapid, stable responses that were linear functions of the logarithm of sodium activity. They were tested under conditions approximating those to be expected in the cell interior, and the results indicated that they can be used to measure intracellular sodium activity without significant interference from intracellular potassium.

Animals↗

Tip potential of open-tip glass microelectrodes: theoretical and experimental studies.

A mathematical analysis of the tip potential based on the main physicochemical phenomena occurring at the tip of a glass microelectrode is presented. The factors considered in the theoretical analysis are the diffusion of ions through the open tip, the conduction in the bulk solutions, the longitudinal conduction in the double layers at the glass-electrolyte interfaces, and to some extent, in a hydrated glass layer. A graphical analysis of the mathematical expressions as a function of the resistivity of test solutions is done and the distribution of the source potentials giving rise to the tip potential is studied. The experimental results presented in the paper confirm the validity of the proposed theoretical model. Comments for an improved use of glass microelectrodes in electrophysiological experiments are given throughout the paper.

Diffusion↗

Tip size of ion-exchanger based K+-selective microelectrodes. II. Effects on measurement of evoked [K+]0 transients.

Double-barreled ion-exchanger based K+-selective microelectrodes (K+ ISMs) of a variety of tip diameters were used to provide an experimental test of predictions that microelectrode tip induced tissue damage influences the magnitude of measured [K+]0 increases. The measured magnitude of stimulation induced [K+]0 rises in the rat optic nerve depended on the tip diameter of the measuring K+ ISM; smaller tipped K+ ISMs tended to measure larger rises of [K+]0 than bigger tipped K+ ISMs. When stimulated [K+]0 increases were simultaneously recorded with 2 K+ ISMs of different tip size, the smaller tipped K+ ISM recorded [K+]0 increases that were, on average, 14% larger than the increases measured by the bigger tipped K+ ISM. In view of these results, and those presented in the previous paper, investigators should standardize the tip sizes of the ISMs used in their experiments.

Action Potentials↗

Controlled bending of high-resistance glass microelectrodes.

The short working distance of compound microscope lenses in many cases prevents microelectrode penetrations of cells perpendicular to the cell surface. Bending electrodes very near their tips removes this constraint. A method is described for bending glass microelectrodes with a hot filament while their tips are immersed in a water drop. Immersion protects the fine electrode tips from the heat and provides control over the angle through which the electrodes are bent.

Electrophysiology↗

Salivary gland K+ transport: in vivo studies with K+-specific microelectrodes.

Stimulation-induced transport of K+ in the submandibular salivary gland of cats and dogs anesthetized with pentobarbital was studied with an extracellular K+-specific microelectrode. Electrical stimulation of the para-sympathetic chorda-lingual nerve caused a rapid transient increase in extracellular K+ concentration from 2.2 to 18.7 meq/liter in the cat and from 2.3 to 15.2 meq/liter in the dog. Eventually the K+ concentration fell below the prestimulatory level, indicating uptake of K+ by the gland cells. In case of prolonged stimulation (2-10 min), the uptake began during stimulation. However, a further reduction in extracellular K+ concentration occurred upon cessation of stimulation, a result that demonstrated that the cells did not fully recover their K+ ,content during stimulation. The latency of the release of K+, defined as the time from the beginning of stimulation to the point at which, the K+-specific microelectrode signal had increased by 2 mV, was 0.6 s in the cat and 0.8 s in the dog. Because these are overestimates of the "true" latencies, we conclude that the K+ release begins simultaneously with the hyperpolarization of the acinar cell membrane.

Animals↗

Microelectrode measurements of K+ and pH in rabbit gastric glands: effect of histamine.

Conventional and liquid ion-exchange microelectrodes sensitive to K+ or pH were used to examine the response of isolated rabbit gastric glands to histamine. The epithelial cells were impaled across the basolateral membrane. The membrane potential averaged -6.1 +/- 0.6 mV and was unchanged after replacement of medium K+, Cl-, or Na+. The intracellular K+ activity (alpha iK) averaged 41.3 +/- 3.0 mM, indicating K+ accumulation by a factor of 6.8. Active accumulation of K+ was eliminated by ouabain. In contrast, histamine increased K+ activity to 55.3 +/- 3.9 mM. This stimulation was blocked by ouabain. In glands bathed in a Na+-free medium containing ouabain, addition of histamine elevated alpha iK from 12.5 +/- 0.7 to 17.1 +/- 1.1 mM. Isobutylmethylxanthine (10(-4) M) also elevated alpha iK. When impaled with pH-sensitive microelectrodes, glands exposed to histamine exhibited regions of acidity as low as pH 3. Acidification was also produced by histamine after medium Na+ had been replaced with choline. Picoprazole (H 149/94) blocked the effects of histamine on alpha iK and gland pH. The results are consistent with the view that histamine-induced acid secretion by gastric glands is associated with K+ uptake by a mechanism that is independent of Na+ transport but is inhibited by intracellular Na+. This is most likely the H+-K+-ATPase on the secretory surface of the gland cells. Evidence that some tissue K+ is bound or compartmentalized is also discussed.

1-Methyl-3-isobutylxanthine↗

Microelectrode studies of Necturus antral mucosa. II. Equivalent circuit analysis.

Intracellular microelectrode techniques were employed to determine the equivalent circuit parameters in Necturus antral mucosa. Stable intracellular impalements were obtained using 15- to 50-M omega microelectrodes. Measured transepithelial and cellular potentials and voltage deflections produced by transepithelial current pulses were used to calculate the electrical resistances of the cell membranes and the equivalent electromotive forces (EMF) at both cell borders. The measured potentials were -4.1 +/- 0.8 mV for the entire epithelia, -41.8 +/- 5.1 mV for the apical membrane, and -45.9 +/- 5.0 mV for the basolateral membrane. Values for the resistances were 7,300 +/- 1,900 omega X cm2 for the apical, 3,990 +/- 1,170 omega X cm2 for the basolateral, and 710 +/- 40 omega X cm2 for the shunt. Assuming that the shunt EMF is zero with control Ringer solution on both sides of the tissue, the effect of this relatively low-resistance shunt on electrical parameters can be determined. The cell membrane EMFs are both oriented with the interior negative and are -1.2 +/- 9.7 mV (apical) and -69.7 +/- 11.3 mV (basolateral). The difference between these values and the measured potentials is the result of a flow of current through the shunt from serosa to mucosa, hyperpolarizing the apical and depolarizing the basolateral membranes.

Amiloride↗

Dual-path capacitance compensation network for microelectrode recordings.

Commercially available microelectrode preamplifiers have a built-in capacity compensation circuit. When a shielded cable is utilized to transmit the signal from the microelectrode to the preamplifier the compensative ability of this circuit may be inadequate and may result in a less than desired frequency response. We describe a dual-path capacitance compensation circuit that resolves this problem without impairment of the shielding properties.

Electronics, Medical↗