Detection of extracellular calcium gradients with a calcium-specific vibrating electrode

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase- sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.     

Electrophysiological responses in guinea pig cochlea to low frequency sound stimuli: distortion of cochlear microphonic (CM) wave form

The present experiment investigated whether or not auditory responses of the middle and/or inner ear in guinea pigs to low frequency sound stimuli [ 60 Hz-2 kHz at 90-120 dB(SPL) ] exhibited the harmonic distortion phenomenon resulting from cochlear microphonics (CM). Measurement of CM leading in turn I by the differential electrode recording method involved measurement of 50 microV isopotential responses, output voltages and CM wave form distortion at each constant sound pressure. The results obtained were as follows: (1) On the 50 microV isopotential response curve and the output voltage curves, the changes at 60-90 Hz were different from those at higher frequencies. (2) At stimuli of 90 or 100 dB(SPL), CM wave form distortion appeared frequently at frequencies below 120 Hz, but were less pronounced above approximately 200 Hz. (3) When raised to 110 and 120 dB(SPL), almost all CM wave forms were distorted at all test frequencies between 60 and 500 Hz. (4) The patterns of CM wave form distortion at frequencies below approximately 120 Hz showed peak clipping and triangular wave distortions, while those at frequencies above approximately 200 Hz showed little of these distortions.     

Interaction of apical and basal membrane ion channels underlies electroreception in ampullary epithelia of skates.

The exquisite sensitivity of elasmobranch fishes to electric fields is thought to reside in electroreceptive organs called ampullae of Lorenzini. We measured the stimulus-response behavior of ampullary organs excised from skates. Under open-circuit conditions, the ampullary organ showed three distinct response states: spontaneous repetitive spikes, evoked spikes, and small, damped oscillatory responses. Under short-circuit conditions, the amplitude range for a linear current response to a sinusoidal (0.5 Hz) voltage clamp of an organ (assessed by spectral analysis of the harmonics generated) was 7-200 microV rms. Changes in the spike firing rate of the afferent nerve innervating the organ were evident for voltage clamps of the ampullary epithelium of 3 microV and the spike rate saturated for clamp steps exceeding 100 microV. Thus, the linear response range of the ampullary epithelium exceeded the range in spike firing rate of the afferent nerve. The steady-state transorgan electrical properties under voltage clamp conditions were obtained by analysis of complex admittance determinations in the frequency range 0.05-20 Hz for perturbations (< 100 microV rms) in the linear range. Admittance functions were distinctly related to the preparation states observed under open-circuit conditions. A negative real part in the organ admittance (i.e., a steady-state negative conductance generated by the preparation) was a common characteristic of the two (open-circuit) excitable states. The negative conductance was also confirmed by the direction of current flow through the ampullary epithelium in response to step voltage clamps.(ABSTRACT TRUNCATED AT 250 WORDS)     

稳态白噪声对豚鼠听觉脑干诱发反应(ABR)功率谱的影响

本文对豚鼠125和131dBSPL稳态白噪声暴露后听觉脑干诱发反应(ABR)功率谱进行了研究.研究结果表明:125dBSPL噪声暴露组0—200、210—500Hz频段谱能量比对照组相应频段谱能量增多(P<0.05),而510—900Hz频段谱能量却显著下降(P<0.01);131dBSPL噪声暴露组0-200Hz频段谱能量显著下降(P<0.01),但210-500Hz、910-1400Hz频段谱能量却明显增多(P<0.01).     

Minimally invasive silicon probe for electrical impedance measurements in small animals

It is commonly accepted that electrical impedance provides relevant information about the physiological condition of living tissues. Currently, impedance measurements are performed with relatively large electrodes not suitable for studies in small animals due to their poor spatial resolution and to the damage that they cause to the tissue. A minimally invasive needle shaped probe for electrical impedance measurements of living tissues is presented in this paper. This micro-probe consists of four square platinum electrodes (300 microm x 300 microm) on a silicon substrate (9 mm x 0.6 mm x 0.5 mm) and has been fabricated by using standard Si microelectronic techniques. The electrodes are not equally spaced in order to optimise the signal strength and the spatial resolution. Characterisation data obtained indicate that these probes provide high spatial resolution (measurement radius <4 mm) with a useful wide frequency band going from 100 Hz to 100 kHz. A series of in vivo experiments in rat kidneys subjected to ischemia was performed to demonstrate the feasibility of the probes and the measurement system. The impedance modulus and phase were measured at 1 kHz since this frequency is sufficiently low to permit the study of the extracellular medium. The extracellular pH and K+ were also simultaneously measured by using commercial miniaturised Ion Selective Electrodes. The induced ischemia period (45 min) resulted in significant changes of all measured parameters (Delta/Z/ approximately 65%; DeltapH approximately 0.8; DeltaK+ approximately 30 mM).     

Respiratory transfer impedance and derived mechanical properties of conscious rats.

A setup is described for measuring the respiratory transfer impedance of conscious rats in the frequency range 16-208 Hz. The rats were placed in a restraining tube in which head and body were separated by means of a dough neck collar. The restraining tube was placed in a body chamber, allowing the application of pseudorandom noise pressure variations to the chest and abdomen. The flow at the airway opening was measured in a small chamber connected to the body chamber. The short-term reproducibility of the transfer impedance was tested by repeated measurements in nine Wistar rats. The mean coefficient of variation for the impedance did not exceed 10%. The impedance data were analyzed using different models of the respiratory system of which a three-coefficient resistance-inertance-compliance model provided the most reliable estimates of respiratory resistance (Rrs) and inertance (Irs). The model response, however, departed systematically from the measured impedance. A nine-coefficient model best described the data. Optimization of this model provided estimates of the respiratory tissue coefficients and upper and lower airway coefficients. Rrs with this model was 13.6 +/- 1.0 (SD) kPa.l-1.s, Irs was 14.5 +/- 1.3 Pa.l-1.s2, and tissue compliance (Cti) was 2.5 +/- 0.5 ml/kPa. The intraindividual coefficient of variation for Rrs and Irs was 11 and 18%, respectively. Because most of the resistance and inertance was located in the airways (85 and 81% of Rrs and Irs, respectively), the partitioning in tissue and upper and lower airway components was rather poor. Our values for Rrs and Irs of conscious rats were much lower and our values for Cti were higher than previously reported values for anesthetized rats.     

Frequency dependent and surface characterization of DNA immobilization and hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued.     

Operational amplifier with adjustable frequency response.

The authors describe an operational amplifier with an adjustable frequency response and its use in membrane physiology, using the voltage clamp and current clamp method. The amplifier eliminates feedback poles causing oscillation. It consists of a follower with a high input resistance in the form of a tube and of an actual amplifier with an adjustable frequency response allowing the abolition of clicks by one pole and of oscillation by two poles in the 500 Hz divided by infinity range. Further properties of the amplifier: a long-term voltage drift of 1 mv, a temperature voltage drift of 0.5 mv/degrees K, input resistance greater than 1 GOhm, amplification greater than 80 dB, output +/- 12 v, 25 ma, noise, measured from the width of the oscilloscope track in the presence of a ray of normal brightness, not exceeding 50 muv in the 0-250 kHz band, f1 = 1 MHz. A short report on the amplifier was published a few years ago (Gulísek and Hencek 1973).     

Voltage responses to tones of outer hair cells in the basal turn of the guinea-pig cochlea: significance for electromotility and desensitization.

Voltage responses were recorded from outer hair cells (OHCS) in the basal coil of the guinea-pig cochlea in response to tones at frequencies above the characteristic frequency (CF) presented together with a 100 Hz tone at 80 dB or 85 dB sound pressure level (SPL). The amplitude and polarity of voltage responses to a 100 Hz, 85 dB SPL tone were altered when presented together with tones at frequencies above CF according to the frequency and level of the high-frequency tone, OHC phasic (ac) (greater than 500 microV) but not tonic (dc) voltage responses were elicited by the high-frequency tone. Thus the responses of OHCS to low-frequency tones can be altered when presented together with a high-frequency tone without an apparent dc change in membrane potential. Recordings were made from an OHC during cochlear desensitization through exposure to an intense tone. The maximum voltage response to high-level low-frequency tones remained unchanged, although the OHC response to high-frequency tones became less sensitive to low-level stimuli and more linear as a function of level. It is suggested that desensitization is associated with a change in the mechanical properties of the cochlea, possibly associated with the OHCS themselves, and not with inactivation of the transducer channels. The amplitude of the OHC ac voltage response was measured at neural threshold, and the consequences of these measurements on hair cell electromotility are considered.     

Impedance electrodes positioned on proximal portions of limbs quantify fluid compartments in dogs

Body resistance and reactance to the conduction of an alternating electrical current were measured using electrodes attached to distal and proximal portions of limbs in anesthetized dogs. Body impedance was calculated from these measurements obtained at 30-min time intervals during a control period and after intravenous administration of 0.9% saline. Extracellular (ECW) and total body water (TBW) were determined by bromide and heavy water dilution techniques, respectively. Baseline impedance obtained from proximal electrodes was related to ECW (r = 0.95, P less than 0.001) and TBW (r = 0.80, P less than 0.02). After saline infusion, proximal electrodes detected a significant fall in impedance (P less than 0.001), whereas distal electrodes did not (P = 0.06). Furthermore, ECW and TBW could be estimated from the drop of proximal impedance after this bolus infusion (r = 0.82, P less than 0.02, and r = 0.86, P less than 0.01, respectively), but not from distal impedance measurements. Proximally placed impedance electrodes are superior to traditionally used distal electrodes for assessment of body fluid changes in the dog.     

Evolution in impedance at the electrode-skin interface of two types of surface EMG electrodes during long-term recordings.

The evolution in impedance at the electrode-skin interface of Beckman and Red Dot electrodes was assessed during long-term recordings. Impedance was measured between each pair of electrodes, arranged in a bipolar configuration on tibialis anterior (n=13). A waveform constructed of sinusoids of known frequencies, evenly distributed on a log scale from 1-16,384 Hz, was applied through the electrodes, and the result recorded by a DAQ system. SEMG signals were recorded at 1000 Hz during isometric dorsiflexion contractions of 30 s, performed every 15 min for 2 h. Impedance data were acquired at 65,536 Hz immediately before and after SEMG recordings. Large individual differences in impedance levels were observed at low frequencies. At high frequencies, impedance values depended only on the electrode type. Impedance decreased steadily with time for Beckman electrodes (p < 0.05), but did not decrease significantly for Red Dot electrodes. The magnitude of the reduction over time varied widely between individuals, and was related to the initial impedance values. The impedance-bandwidth product remained constant for each electrode type (95% confidence intervals 146.2-148.2 and 126.1-127.8 for Beckman and Red Dot electrodes respectively). When skin impedance is electrically modelled with a simple network containing a resistor and a capacitor, the capacitance varies with the properties of the electrode used, whereas resistance is dependent on the subject. Furthermore, the EMG spectrum is unaffected by impedance provided skin preparation is sufficient to reduce the impedance below 55 komega.     

An endothelial cell compatible biosensor fabricated using optically thin indium tin oxide silicon nitride electrodes

This study describes the fabrication and performance of an endothelial cell compatible, optically thin, indium tin oxide (ITO) microimpedance biosensor. The biosensor was constructed by sputtering a thin insulating layer of silicon nitride (Si(3)N(4)) onto a 100 nm thick ITO layer. Indium tin oxide electrodes were formed by chemically etching 250 or 500 microm diameter holes through the Si(3)N(4) insulating layer. The exposed ITO electrode was electrically connected to an ITO counter electrode, approximately 2 cm(2) in area, via a 400 microL well containing cell culture media. A lock-in amplifier circuit monitored the impedance of porcine pulmonary artery endothelial cells (PPAECs) cultivated on the electrodes as a function of frequency, between 10 and 100 kHz, and as a function of time, at 5.62 kHz. The ITO-Si(3)N(4) microelectrodes provided consistent and repeatable impedance measurements to the attachment and spreading of PPAECs. In addition, the ITO-Si(3)N(4) electrodes were recyclable, robust, resistant to ethanol sterilization, and had a high optical transmittance. Most importantly, the ITO-Si(3)N(4) electrodes allowed optical access for dynamic cellular attachment imaging. The 5.62 kHz time dependent cellular impedance response to the drug Cytochalasin D further demonstrated the feasibility of using this electrode configuration for dynamic cellular impedance studies.     

Electrical frequency dependent characterization of DNA hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of electrochemical impedance spectroscopy (EIS) and quartz crystal microgravimetry (QCM). Synthetic 5'-amino terminated single stranded oligonucleotides (ssDNA) were attached to the exposed glass surface between the digits of microlithographically fabricated interdigitated microsensor electrodes using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA immobilization was achieved to the surface of the gold driving electrodes of AT-cut quartz QCM crystals using 3-mercaptopropyl-trimethoxysilane. Significant changes in electrochemical impedance values (both real and imaginary components) (11% increase in impedance modulus at 120 Hz) and resonant frequency values (0.004% decrease) were detected as a consequence of hybridization of the bound ssDNA upon exposure to its complement under hybridization conditions. Non-complementary (random) sequence sowed a modest decrease in impedance and a non-detectable change in resonant frequency. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA, has been demonstrated. The potential for development of label-free, low density DNA microarrays is demonstrated and is being pursued.     

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution.

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.     

Functional organization of the electroreceptive midbrain in an elasmobranch (Platyrhinoidis triseriata)

The response properties of 322 single units in the electroreceptive midbrain (lateral mesencephalic nucleus, LMN) of the thornback ray, Platyrhinoidis triseriata, were studied using uniform and local electric fields. Tactile, visual, or auditory stimuli were also presented to test for multimodality. Most LMN electrosensory units (81%) are silent in the absence of stimulation. Those with spontaneous activity fired irregularly at 0.5 to 5 impulses/s, the lower values being more common. Two units had firing rates greater than 10/s. Midbrain electrosensory units are largely phasic, responding with one or a few spikes per stimulus onset or offset or both, but the adaptation characteristics of some neurons are complex. The same neuron can exhibit phasic or phasic-tonic responses, depending upon orientation of the electric field. Tonic units without any initial phasic over-shoot were not recorded. Even the phasic-tonic units adapt to a step stimulus within several seconds. Unit thresholds are generally lower than 0.3 microV/cm, the weakest stimulus delivered, although thresholds as high as 5 microV/cm were recorded, Neuronal responses reach a maximum, with few exceptions, at 100 microV/cm and decrease rapidly at higher intensities. LMN neurons are highly sensitive to stimulus repetition rates: most responded to frequencies of 5 pulses/s or less; none responded to rates greater than 10/s. Three distinct response patterns are recognized. Best frequencies in response to sinusoidal stimuli range from 0.2 Hz (the lowest frequency delivered) to 4 Hz. Responses decrease rapidly at 8 Hz or greater, and no units responded to frequencies greater than 32 Hz. Most LMN neurons have small, well defined excitatory electroreceptive fields (RFs) exhibiting no surround inhibition, at least as detectable by methods employed here. Seventy-eight percent of units recorded had RFs restricted to the ventral surface: of these, 98% were contralateral. The remaining 22% of units had disjunct dorsal and ventral receptive fields. Electrosensory RFs on the ventral surface are somatotopically organized. Anterior, middle, and posterior body surfaces are mapped at the rostral, middle, and caudal levels, respectively, of the contralateral LMN. The lateral, middle, and medial body are mapped at medial, middle, and lateral levels of the nucleus. Moreover, the RFs of all units isolated in a given dorsoventral electrode track are nearly superimposable. About 40% of LMN, measured from the dorsal surface, is devoted to input from ventral electroreceptors located in a small region rostral and lateral to the mouth.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metallic oxide CdIn2O4 films for the label free electrochemical detection of DNA hybridization

DNA functionalised semiconductor metallic oxide electrodes have been developed for the direct electrochemical detection of DNA hybridization, without labelling or the introduction of a redox couple. Conductive CdIn(2)O(4) thin films with controlled properties were deposited on glass substrates using an aerosol pyrolysis technique. The films exhibit a polycrystalline microstructure with a surface roughness of 1.5 nm (r.m.s.) and an electrical resistivity ranging between 1 and 3 x 10(-3) Omega cm. These electrodes were functionalised using hydroxylation and silanisation steps, to allow the binding of DNA probe sequences (20 bases). The electrical detection of DNA hybridization with complementary sequences has been performed using electrochemical impedance spectrometry (EIS) measuring the variation of impedance before and after hybridization. Two set-ups were used, a standard set-up including three electrodes and a set-up including two symmetrical electrodes. In both configurations, a significant increase of the impedance modulus, more particularly of the real part of the impedance (160-225% according to the electrochemical cell used) has been obtained over a frequency range of 10-10(5)Hz. DNA hybridization has also been systematically confirmed using the fluorescence spectrometry. This study emphasizes the high sensitivity of the CdIn(2)O(4) as a working electrode for the detection of biological events occurring at the electrode surface.     

Localization and function of the electrical oscillation in electroreceptive ampullary epithelium from skates.

A steady, spontaneous current oscillation (1 nA p-p) occurs in voltage-clamped, isolated ampullary organs (canal, ampulla, and nerve) from skates (Raja). Spectral analysis showed that energy in the oscillation was confined to a narrow band of frequencies (3 Hz) about a fundamental frequency (32 Hz at 20 degrees C) and in harmonics. The frequency of the oscillation was temperature dependent (increasing from 21 to 33 Hz for increases in temperature from 13 degrees C to 21 degrees C). The addition of 0.5 microM tetrodotoxin to the basal side of the ampullary epithelium eliminated afferent nerve activity but had no effect on the epithelial oscillation, indicating that the oscillation is not generated or induced by afferent nerve activity. Nitrendipine (2 microM) added to the solution bathing the basal side of the ampullary epithelium abolished the oscillation rapidly (within minutes), but a steady-state negative conductance (i.e., real part of the complex admittance < 0) generated by the preparation remained for 36 min. Conversely, nitrendipine (50 microM) added to the perfusate (artificial sea water) of the apical side eliminated the negative conductance rapidly (18.5 min) but had no effect on the spontaneous oscillation for more than 1 h. The effect and the elapsed time for an effect of nitrendipine after separate applications to the basal and apical membrane surfaces of ampullary epithelium suggest that 1) the negative conductance and the oscillation are generated independently in apical and basal membranes, respectively, and 2) both processes involve L-type Ca channels. Furthermore, the addition of tetraethylammonium (2 mM) to the basal side eliminated both the oscillation and the postsynaptic response to voltage clamps (< or = 100 microV) of the ampullary epithelium in the operational voltage range of the afferent nerve. This result suggests that the basal membrane oscillation functions in neurotransmitter release from presynaptic (basal) membranes.     

Impedance profiles of peripheral and central neurons

The electrical impedance of trigeminal ganglion cells (in vivo) and hippocampal CA1 neurons (in vitro) of guinea pigs was measured in the frequency range of 5-1250 Hz using intracellular recording techniques with single microelectrodes and computerized methodology. The transfer functions of the electrode and the electrode-neuron system were computed from the ratio of fast Fourier transforms of the output voltage response from the neuron and input current composed of sine waves with rapidly increasing frequency which displaced membrane potential by 2-5 mV. We believe these to be the first measurements of complex impedance and transfer functions in peripheral and central neurons of vertebrates and the first use of such input current functions. The majority of trigeminal ganglion cells did not exhibit electrical behaviour ascribable to a simple resistance-capacitance (RC) circuit but showed a hump at low frequencies (5-250 Hz) in the computed transfer function, probably attributable to resonance. The transfer function in less than 20% of the trigeminal neurons could be fitted approximately to a theoretical transfer function (resistance in series with a parallel RC circuit model) providing values for electrode resistance, effective input resistance, and effective input capacitance. The transfer functions measured in hippocampal CA1 neurons were characterized by a rapid fall-off in the low frequency range (less than 200 Hz). Impedance locus plots approximate the locus corresponding to a series RC circuit in parallel with a parallel RC circuit.