Ceramic-based multisite microelectrode arrays for simultaneous measures of choline and acetylcholine in CNS

A ceramic-based microelectrode array (MEA) with enzyme coatings for the accurate measurement of acetylcholine (ACh) in brain tissues is presented. Novel design features allow for self-referencing recordings for improved limits of detection and highly selective measurements of ACh and choline (Ch), simultaneously. Design and fabrication features also result in minimal tissue damage during implantation and improved enzyme coatings due to isolated recording sites. In these studies we have used a recombinant human acetylcholinesterase enzyme coating, which has better reproducibility than other commercially available enzymes. The precisely patterned recording site dimensions, low limit of detection (0.2 micro M) and fast response time ( approximately 1s) allow for second-by-second measurements of ACh and Ch in brain tissues. An electropolymerized meta-phenylenediamine (mPD) layer was used to exclude interfering substances from being recorded at the platinum recording sites. Our studies support that the mPD layer was stable for over 24h under in vitro and in vivo recording conditions. In addition, our work supports that the current configuration of the MEAs produces a robust design, which is suited for measures of ACh and Ch in rat brain.     

MRI-based localization of electrophysiological recording sites within the cerebral cortex at single-voxel accuracy

The localization of microelectrode recording sites in the layers of primate cerebral cortex permits the analysis of relationships between recorded neuronal activities and underlying anatomical connections. We present a magnetic resonance imaging method for precise in vivo localization of cortical recording sites. In this method, the susceptibility-induced effect thickens the appearance of the microelectrode and enhances the detectability of the microelectrode tip, which usually occupies less than a few percent of the volume of an image voxel. In a phantom study, the optimized susceptibility-induced effect allowed tip detection with single-voxel accuracy (in-plane resolution, 50 mum). We applied this method to recording microelectrodes inserted into the brains of macaque monkeys, and localized the microelectrode tip at an in-plane resolution of 150 mum within the cortex of 2-3 mm in thickness. Subsequent histological analyses validated the single-voxel accuracy of the in vivo tip localization. This method opens up a way to investigate information flow during cognitive processes in the brain.     

An improved Na+-selective microelectrode for intracellular measurements in plant cells.

The high background K+ concentration in plant cells is a problem for intracellular measurements of Na+ using ion-selective microelectrodes. The discrimination between Na+ and K+ of the microelectrode ionophore molecule limits the usefulness of this technique. A new Na+-selective microelectrode with an ionophore incorporating a tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene has been developed. Microelectrodes made with this new sensor have superior selectivity for Na+ over K+ resulting in a lower limit of detection when compared with microelectrodes made using a commercially available ionophore (ETH227). Both types of microelectrodes were insensitive to changes in ionic strength and physiological ranges of pH, but only the calixarene-based electrodes showed no protein interference. To test the suitability of the calixarene-based microelectrodes for measurements in plants, they were used to measure Na+ in epidermal cells in the zone 10-20 mm from the root apex of barley (Hordeum vulgare L.). Seedlings were grown in a nutrient solution containing 200 mM NaCl for 1-6 d. The range of intracellular Na+ activity (a(Na)) measured varied from < or =0.1 mM (limit of detection) to over 100 mM, and these values increased significantly with time. The membrane potential (E(m)) of these cells was variable, but the values became significantly more negative with time, although there was no significant correlation between E(m) and a(Na). These intracellular measurements could not be separated into distinct populations that might be representative of subcellular compartments.     

Increases in cholinergic neurotransmission measured by using choline-sensitive microelectrodes: enhanced detection by hydrolysis of acetylcholine on recording sites?

Previous experiments demonstrated that second-based transient increases in choline concentrations measured by electrodes coated with choline oxidase (ChOx) and the amperometric detection of hydrogen peroxide validly indicate the depolarization-dependent release of acetylcholine (ACh) and its hydrolysis by endogenous acetylcholinesterase (AChE). Therefore, choline-sensitive microelectrodes have become valuable tools in neuropharmacological and behavioral research. The present experiments were designed to test the possibility that co-immobilization of ChOx plus AChE on recording sites increases the level of detection for evoked ACh release in the brain. If newly released ACh is not completely hydrolyzed by endogenous AChE and capable of reaching the extracellular space, currents recorded via sites equipped with both enzymes should be greater when compared with sites coated with ChOx only. Pairs of platinum-recording sites were coated either with AChE plus ChOx or ChOx alone. Potassium or nicotine-evoked currents were recorded throughout the entire dorsal–ventral extent of the medial prefrontal cortex (mPFC). The amplitudes of evoked cholinergic signals did not differ significantly between AChE + ChOx and ChOx-only coated recording sites. Additional experiments controlling for several potential confounds suggested that, in vivo, ACh levels ≥150 fmol were detected by recordings sites featuring dual enzyme coating. Collectively, these results indicate that co-coating of microelectrodes with AChE does not enhance the detection of cholinergic activity in the cortex compared with measurements via recording sites coated only with ChOx.     

Comparing cardiac action potentials recorded with metal and glass microelectrodes

Machine-pulled high-impedance glass capillary microelectrode is standard for transmembrane potential (TMP) recordings. However, it is fragile and difficult to impale, especially in beating myocardial tissues. We hypothesize that a high-impedance pure iridium metal electrode can be used as an alternative to the glass microelectrode for TMP recording. The TMPs were simultaneously recorded from isolated perfused swine right ventricles with a metal microelectrode and a standard glass microelectrode during pacing and during ventricular fibrillation. The basic morphology of TMP recorded with these electrodes was comparable. The action potential duration (APD) at 90% repolarization was 241 +/- 29 ms for the metal microelectrode and 236 +/- 31 ms for the glass microelectrode with a good correlation (r = 0.99, P < 0.0001). The maximum slope value of the APD restitution curves during pacing was also significantly correlated. One metal microelectrode and >20 glass microelectrodes were needed per study. We conclude that, in isolated perfused swine right ventricles, the TMP recorded by the metal microelectrode is comparable with that recorded by the glass microelectrode. Because the metal microelectrode is more durable than the glass microelectrode, it can serve as an alternative for APD recording and for restitution analyses.     

Electrochemical Properties of Hydrated Cation-Selective Glass Membrane: A Model of K+ and Na+ Transport

Electrochemical properties of cation-selective glass microelectrodes made from NAS_27-04 were studied. There was a marked fall in electrical resistance of the microelectrodes stored in 3 M KCl solution (aging). The resistance was in the range of 2 × 10⁷ to 10⁹ Ω, which were much lower than those estimated from the electrical resistivity of dry glass for the equivalent dimensions of microelectrode working tips. This fall in resistance was accompanied by an increase in microelectrode selectivity for K⁺. The low resistance and increased K⁺ selectivity are desirable features that make the microelectrode more suitable for application to biologic studies. The changes in microelectrode resistance and selectivity were interpreted to be due to hydration of the entire thickness of the glass membrane, resulting in a change in the field strength of anionic sites and formation of ionic channels in the glass membrane. Thus, the fall in resistance is explained by decrease in energy barrier, which is equivalent to the activation energy of interaction between the cations and anionic sites in the glass membrane. Some of the microelectrodes showed a transient depolarization that resembled the action potential of a biological membrane. This transient depolarization was associated with the changes in microelectrode resistance and selectivity. The transient depolarizations suggest the temporary development of wide channels in the membrane permitting free movement of hydrated cations according to the bulk electrochemical gradient.     

Cortical choline transporter function measured in vivo using choline-sensitive microelectrodes: clearance of endogenous and exogenous choline and effects of removal of cholinergic terminals

The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.     

Simultaneous Measurement of Intracellular pH and K+ or NO3- in Barley Root Cells Using Triple-Barreled,Ion-Selective Microelectrodes

The manufacture and use of triple-barreled microelectrodes, which are capable of simultaneous in vivo measurement of intracellular pH and the activities of K+ or NO3- and cell membrane potential (Em), are described. Scanning electron micrographs showed that the three tips were aligned and that the overall tip diameter was approximately 0.8 [mu]m. When filled with 100 mM KCl, all three barrels simultaneously reported identical transmembrane potentials, showing that all three tips were located in the same subcellular compartment. Intracellular estimates of Em in barley (Hordeum vulgare L. cv Klaxon) root epidermal cells obtained with these triple-barreled microelectrodes were indistinguishable from those obtained using single- or double-barreled microelectrodes. Measurements made with triple-barreled K+ and pH-selective microelectrodes in root cells of 7-d-old barley plants grown in a nutrient solution containing 0.5 mM K+ yielded cytosolic and vacuolar populations having mean K+ activity values of 71.3 and 68.7 mM, respectively. The associated mean pH values ([plus or minus]SE) were 7.26 [plus or minus] 0.06 (cytosol) and 5.18 [plus or minus] 0.08 (vacuole). Analysis of whole-tissue digests confirmed the microelectrode measurements. Measurements made using triple-barreled pH- and nitrate-selective microelectrodes confirmed earlier double-barreled measurements of pH and nitrate in barley root epidermal cells growing in 10 mM nitrate.     

Use of micromachined probes for the recording of cardiac electrograms in isolated heart tissues

Micromachined probes, with iridium (Ir) microelectrodes on silicon shanks, were evaluated to assess their suitability for cardiac electrogram recording. The electrochemical activation (anodic oxidation) procedure for the circular Ir microelectrode was investigated using the square wave potential according to the electrode size, number of cycles, and cathodic-anodic potential level of the square wave. Increase in the charge storage capacity was pronounced either in smaller electrodes or with higher potential level of the square wave. The electrode impedance reduced in a similar manner with increasing number of cycle irrespective of the electrode size. With either lower potential level (-0.70/+0.60 V) or smaller number of cycle (200 cycles) than those for the activation of stimulating electrode, the likelihood of overactivation of the recording microelectrode can be minimized. These anodic IrOx film (AIROF) microelectrodes were used for the recording of extracellular electrograms in two different ex vivo cardiac tissue preparations. A single-shank microprobe was applied to the left ventricle of a mouse heart. Both the spontaneous and paced transmural responses propagating between epicardium and endocardium were obtained. Longitudinal cardiac wavefronts propagating along the rabbit papillary muscle were also recorded with a unique multiple-shank design. The measured mean amplitude and the propagation velocity of the extracellular voltage were 12.2 +/- 1.8 mV and 58.9 +/- 2.2 cm/s, respectively (n = 27). These microprobes with precisely defined electrode spacing make a useful tool for the spatial and temporal mapping of electrical properties in isolated heart tissues ex vivo.     

Influence of the first amplifier stage in MEA systems on extracellular signal shapes

Recording of extracellular signals with planar metal microelectrodes (ME) has already been presented more than 30 years ago. To date, microelectrode array (MEA) systems are able to measure extracellular signals at about 64 sites, simultaneously. This enables monitoring of electrical activity of many cells in a large area. The extracellular recording technique has become a widely used method for neurological, toxicological or pharmacological studies. It already proved its potential to supplement the classical methods in electrophysiology. The interpretation of the recorded signal shapes in order to extract electrophysiological meaningful data--however--is still under discussion. In this article, we analyse the preamplifier circuit for extracellular recording of cardiac myocyte signals. We use a circuit model for the cell-electrode contact including the first amplification stage. In test experiments, we observe different signal shapes, when different shunt resistors are introduced at the input of the preamplifier. According to the frequency spectra of the recordings, we evaluate the transfer function between the source signal and the readout signal. As a result of our studies, an optimum readout electronics for originally, preserved extracellular signal shapes is proposed. Our amplifier design will be most valuable, if the use of small microelectrodes with high input impedances for in vitro as well as for in vivo experiments is desired.     

Intracellular chloride activity in rabbit papillary muscle: effect of serum

The intracellular chloride activity (aiCl), measured with Cl-selective microelectrodes on stimulated rabbit papillary muscles (1 Hz) incubated in serum, was 7.2 +/- 2.2 mM (48 measurements). Under the same condition, on the quiescent muscle, aiCl was 7.5 +/- 2.8 mM (45 measurements). The membrane potential of quiescent papillary muscles and diastolic potential of stimulated papillary muscles were -79.0 +/- 0.7 (50 measurements) and -83.5 +/- 0.5 mV (50 measurements), respectively. The experimental conditions were chosen to reproduce the in vivo conditions where the Cl equilibrium potential is close to the membrane potential or to the diastolic potential. After correcting for cytoplasmic interference (4 mM) on the aiCl measurements, the Cl equilibrium potential (ECl) was -84 mV. In conclusion, the Cl distribution in cardiac cells bathed in serum is passive as for in vivo cardiac cells.     

Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.     

Measurement of the Cytoplasmic pH in Nitella translucens: Comparison of Values Obtained by Microelectrode and Weak Acid Methods

A comparison has been made between the use of two types of pH microelectrode and the weak acid method for determining the cytoplasmic pH of Nitella translucens at an external pH of 6. There was good agreement between the value obtained with glass pH microelectrodes (7.54 +/- 0.15 se) and that obtained using the weak acid 5,5-dimethyloxazolidine-2,4-dione (7.42 +/- 0.07 se). Plastic-insulated antimony microelectrodes gave a significantly lower value (6.74 +/- 0.15 se) possibly due to disruption of the insulation by the cell wall. The addition of 1 mM NaN(3) rapidly reduced the pH recorded by the glass pH microelectrodes to about 5.3. A smaller change was observed using the weak acid method. The relevance of this observation to recent work on indoleacetic acid transport is discussed.     

Effects of membrane depolarization and divalent cations on anaphylactic histamine secretion

The effects of membrane depolarization and divalent cations on histamine release have been studied in sensitized mast cells. Membrane potential of these cells has been measured with intracellular microelectrodes. Our results show that mast cells have a large resting potential (-61 +/- 12 mV) however they do not generate active membrane electrical responses when are depolarized by passing current through the recording microelectrode. High external K+ does not increase histamine release. Histamine secretion is supported by alkali-earth divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) but strongly inhibited by transition metals. Ca2+ concentrations above 1 mM inhibit histamine release, however, this effect is not mimicked by Sr2+ and Ba2+.     

Gold nanograin microelectrodes for neuroelectronic interfaces

Neuroelectronic interfaces are imperative in investigating neural tissues as electrical signals are the main information carriers in the nervous system and metal microelectrodes have been widely used for recording and stimulation of nerve cells. For high performance microelectrodes, low tissue-electrode interfacial impedance and high charge injection limits are essential and nanoscale surface engineering has been utilized to meet the requirements for microelectrodes. We report a single-cell sized microelectrode, which has unique gold nanograin structures, using a simple electrochemical deposition method. The fabricated microelectrode had a sunflower shape with 1–5 (m of micropetals along the circumference of the microelectrode and 500 nm nanograins at the center. The nanograin electrodes had 69-fold decrease of impedance and 10-fold increase in electrical stimulation capability compared to unmodified flat gold microelectrodes. The recording and stimulation performance of nanograin electrodes was tested using dissociated rat hippocampal neuronal cultures. Noise levels were extremely low (2.89 μV_rms) resulting in high signal-to-noise ratio for low-amplitude action potentials (18.6–315 μV). Small biphasic current pulses (20–60 μA) could evoke action potentials from neurons nearby electrodes. This new nanostructured neural electrode may be applicable for the development of cell-based biosensors or clinical neural prosthetic devices.     

Qualitative measurements of the entry of L-lactate into single surface fibres of frog skeletal muscle using a lactate-sensitive microelectrode

The present results demonstrate the sensitivity of the Corning chloride liquid ion exchanger 477913 to L-lactate. Microelectrodes filled with this exchanger showed responses to changes in L-lactate concentration in chloride-free solutions. In these experiments L-lactate replaced gluconate in equimolar amounts. Microelectrodes filled with this exchanger were used to qualitatively detect changes in intracellular anion in chloride-depleted frog sartorius muscle fibres during exposure to extracellular concentrations of L-lactate. The increase in intracellular anion concentration is consistent with the movement of L-lactate into the cell. This microelectrode enables one to qualitatively monitor changes in intracellular L-lactate in chloride-free experiments without incorporating selectivity coefficients.     

Design of ionophores for ion-selective microsensors

Requirements for a reliable use of liquid membrane microelectrodes are discussed in terms of stability, response time, and lifetime on the basis of membrane technological considerations. The selectivity of H+, Li+, Na+, K+, Mg2+, Ca2+, and Cl- microelectrodes is critically evaluated using the Nikolskii-Eisenman formalism. Recent progress in the design of new ionophores is presented. A novel neutral carrier-based Ca2+-selective microelectrode with a detection limit of about 5 X 10(-10) M Ca2+ at a background of 125 mM K+ has been realized. An neutral carrier-based microelectrode for H+ with extended pH range of the sample solution is now available. Promising developments in the field of Li+-, Mg2+-, and Cl--selective ionophores are discussed.     

Sonochemically fabricated microelectrode arrays for biosensors--part II. Modification with a polysiloxane coating

A polymer-modified sonochemically fabricated glucose oxidase microelectrode array with microelectrode population densities of up to 2.5 x 10(5) microelectrodes per square centimetres is reported. These microelectrode sensors were formed by first depositing an insulating film on commercial screen printed electrodes which was subsequently sonicated to form cavities of regular sizes in the film. Electropolymerisation of aniline at the microelectrode cavities formed polyaniline protrusions containing entrapped glucose oxidase. Chemical deposition of polysiloxane from dichlorodimethysilane was used to deposit a thin protective and diffusion mass transport controlling coating over the electrodes. The physical and electrochemical properties of these films were studied. The performance of the final glucose oxidase based microelectrode sensor array is reported.     

Neutral carrier Na+- and Ca2+-selective microelectrodes for intracellular application.

Na+- and Ca2+-selective microelectrodes were made with Simon's neutral carrier ETH 227 and ETH 1001, respectively, and their properties were studied for intracellular application. The kNaK (selectivity coefficient for Na+ with respect to K+) values of the Na+-selective microelectrodes were in the range of 0.01-0.02, which is comparable to those of recessed-tip Na+-selective glass microelectrodes. The kNaMg values of the microelectrodes were approximately 0.005 so that the interference by intracellular Mg2+ levels could be negligible. The kNaCa values were approximately 2 and the Na+-selective microelectrodes were more selective to Ca2+ than Na+. This indicates that their intracellular application requires special care to handle Ca2+ interference under certain conditions. The kNaK, kNaMg, and kNaCa values did not depend significantly on the methods used for their determination or on the ion activity levels tested. The Nicolsky equation described well the microelectrode potentials in the mixed solutions of NaCl (1-100 mM) and KCl. Potential and resistance of the microelectrodes were stable for a long period and their response time was fast. The results indicate that the Na+-selective microlectrodes are suitable for measurements of intracellular Na ion activities. Ca2+-selective microelectrode potentials at Ca2+ concentrations lower than 10(-4) M changed significantly for the first 2-3 h and then became fairly stable. The rate of the potential change was dependent on the column length of the Ca2+-selective liquid filled. Potentials of the microelectrodes varied from 10-20 mV for Ca2+ between 10(-7) and 10(-6) M concentrations, which may be the cytosolic free-Ca2+ range. With the Ca2+ concentrations greater than 10(-6) M, the microelectrodes had potential changes of approximately 30 mV or greater for a tenfold change in Ca2+ concentration. The kCaK and kCaNa values were in the ranges of 10(-5)-10(-6) and 10(-4)-10(-5), respectively. The kCaMg values were approximately 10(-7). The results show that the Ca2+-selective microelectrodes can be used for measurements of cytosolic Ca ion activities.