Voltage clamping with a single microelectrode.

A technique is described which allows neurons to be voltage clamped with a single microelectrode, and the advantages of this circuit with respect to conventional bridge techniques are discussed. In this circuit, the single microelectrode is rapidly switched from a current passing to a recording mode. The circuitry consists of: (1) an electronic switch; (2) a high impedance, ultralow input capacity amplifier; (3) a sample-and-hold module; (4) conventional voltage clamping circuitry. The closed electronic switch allows current to flow through the electrode. The switch then opens, and the electrode is in a recording mode. The low input capacity of the preamplifier allows the artifact from the current pulse to rapidly abate, after which time the circuit samples the membrane potential. This cycle is repeated at rates up to 10 kHz. The voltage clamping amplifier senses the output of the sample-and-hold module and adjusts the current pulse amplitude to maintain the desired membrane potential. The system was evaluated in Aplysia neurons by inserting two microelectrodes into a cell. One electrode was used to clamp the cell and the other to independently monitor membrane potential at a remote location in the soma.     

Resistivity of axoplasm. II. Internal resistivity of giant axons of squid and Myxicola

The specific resistivity of the axoplasm of giant axons of squid and Myxicola was measured utilizing a single metal microelectrode subjected to alternating current in a circuit in which the voltage output varies with the conductivity of the thin layer of fluid at the exposed electrode tip. The average specific resistivity of stellar axons of Loligo pealei was 31 omegacm (1.55 times seawater [X SW]) while for Loligo opalescens it was 32 omegacm (1.30 X SW). Smaller giant axons had a higher average resistivity. Myxicola giant axons had a resistivity of 68 omegacm (2.7 X SW) in normal seawater, and 53 omegacm (2.1 X SW) in a hypertonic high-Mg++ seawater. The temperature dependence of squid axon resistivity does not differ from that of an equally conductive dilution of seawater.     

The use of metal microelectrodes in broad-band recording.

We checked to what extent the voltage-current relationship of a microelectrode is linear. In the linear domain we measured the impedance characteristic of the electrode. We measured the frequency response curve and the phase characteristic of the electrode and amplifier system.     

Involvement of pedal peptide in locomotion in Aplysia: modulation of foot muscle contractions

Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay (RIA) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends an axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in "split-foot" preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.     

Immunological characterization of an anti-actin antibody specific for cytoplasmic actins and its use for the immunocytological localization of actin in Aplysia nervous tissue

Previous immunochemical and immunocytochemical studies have shown that an antibody to actin prepared from body wall muscle of the marine mollusc Aplysia californica is specific for vertebrate cytoplasmic actins. The ability of this anti-actin to distinguish between different forms of actin most likely reflects the recognition of amino acid sequences unique to cytoplasmic actins. We have confirmed the specificity of this antibody for cytoplasmic actins using nervous tissue as a source of cytoplasmic actin in further immunochemical studies. In addition to binding cytoplasmic actin in purified preparations, the antibody removed actin selectively from crude extracts of nervous tissue of some but not all of the species tested. Our results also suggest that tissue-specific differences in the distribution of cytoplasmic actins may exist. Immunofluorescence studies of Aplysia nervous tissue stained with anti-actin revealed that actin is present in the cell body and axonal processes of Aplysia neurons. Although the function of actin in nerve cells is not understood, the observed pattern of immunofluorescence staining is consistent with the idea that actin may be involved in movement within the axoplasm.     

The effect of dendritic voltage-gated conductances on the neuronal impedance: a quantitative model

Neuronal impedance characterizes the magnitude and timing of the subthreshold response of a neuron to oscillatory input at a given frequency. It is known to be influenced by both the morphology of the neuron and the presence of voltage-gated conductances in the cell membrane. Most existing theoretical accounts of neuronal impedance considered the effects of voltage-gated conductances but neglected the spatial extent of the cell, while others examined spatially extended dendrites with a passive or spatially uniform quasi-active membrane. We derived an explicit mathematical expression for the somatic input impedance of a model neuron consisting of a somatic compartment coupled to an infinite dendritic cable which contained voltage-gated conductances, in the more general case of non-uniform dendritic membrane potential. The validity and generality of this model was verified through computer simulations of various model neurons. The analytical model was then applied to the analysis of experimental data from real CA1 pyramidal neurons. The model confirmed that the biophysical properties and predominantly dendritic localization of the hyperpolarization-activated cation current I (h) were important determinants of the impedance profile, but also predicted a significant contribution from a depolarization-activated fast inward current. Our calculations also implicated the interaction of I (h) with amplifying currents as the main factor governing the shape of the impedance-frequency profile in two types of hippocampal interneuron. Our results provide not only a theoretical advance in our understanding of the frequency-dependent behavior of nerve cells, but also a practical tool for the identification of candidate mechanisms that determine neuronal response properties.     

Harmonic system analysis of the algae Valonia utricularis: contribution of an electrogenic transport system to gain and phase-shift of the transfer function

Cell membrane properties of the giant marine alga Valonia utricularis were measured in the frequency domain between 1 Hz and 10 MHz by harmonic system analysis. Harmonic analysis was performed by imposing a sinusoidal electrical voltage on the cell interior via an internal microelectrode. Gain and phase-shift of the resulting sinusoidal membrane voltage were measured over the whole frequency range with an internal voltage microelectrode. Bode plots of gain and phase-shift allowed the determination of the electrical parameters of the equivalent electronic circuits of the cell membrane of V. utricularis, which showed dynamic and passive properties dependent on the pH of the external aqueous solution. The dynamic components of the membrane impedance were caused by an electrogenic transport system for chloride described previously (Wang, J., G. Wehner, R. Benz, and U. Zimmermann. 1991. Biophys. J. 59:235-248). The kinetic and equilibrium parameters of the transport system could be evaluated from the fit of Bode plots of gain and phase-shift. The frequency domain technique revealed complete agreement of transport parameters with previously published results. The data demonstrate that an electrogenic transport system can be driven by an oscillating electric field.     

EFFECTS OF NERVE STIMULATION ON THE METABOLISM OF RIBONUCLEIC ACID IN A MOLLUSCAN GIANT NEURONE

—In vitro experiments were performed in order to determine whether nerve stimulation would affect the RNA metabolism of an identified giant neurone (R2) in the abdominal ganglion of Aplysia californica. The electrophysiological activity of the neurone was continuously monitored with an intra- or extracellular microelectrode. The mere presence of an intracellular microelectrode inside the neurone had no significant effect on the incorporation of tritiated nucleosides into the RNA of the giant neurone. Prolonged electrical stimulation of ganglionic nerves, strong enough to elicit post-synaptic spikes in the giant neurone, produced a marked increase in the amount of labelled RNA in the nucleus as well as in the cytoplasm. Electrophoresis studies suggested that this increase in labelling might concern RNA with molecular weights corresponding to ribosomal as well as to non-ribosomal RNA.     

Ability of the Ca2+-selective microelectrodes to measure fast and local Ca2+ transients in nerve cells

The ability of the Ca2+-selective microelectrode to measure fast Ca2+ transients intracellularly is reviewed. In vitro, Ca microelectrodes can respond to Ca2+ injections with time to peaks as small as 40 ms. We present methods to improve the dynamic response of Ca microelectrodes and to make Ca-buffered solutions in high ionic strength. Examples of measurements of intracellular free Ca2+ [( Ca2+]i) transients in Aplysia neurons and in Limulus photoreceptors are shown. To show the validity of those measurements, simultaneous recordings of the Arsenazo III (AIII) absorbance and of the Ca-selective electrode potential were made in voltage-clamped neurons of the abdominal ganglion of Aplysia californica. Pressure injection of AIII to a concentration of 300-500 microM induced a rise in resting [Ca2+]i; injection of higher [AIII] led to buffering of [Ca2+]i transients. Both techniques responded to changes in resting [Ca2+]i in the same direction except that AIII showed an increase in absorbance in 0 [Ca2+]o. Voltage-clamp pulses transiently increased both the AIII absorbance and the Ca2+ electrode potential. Reducing or increasing the driving force for Ca2+ entry changed the magnitude of both signals in the right direction. Examples of spatial localization of [Ca2+]i increases and Ca2+ gradients within the cytoplasm were demonstrated using the Ca electrode. The use of optical techniques to measure local [Ca2+]i changes is briefly reviewed.     

Spatial Analysis of Slowly Oscillating Electric Activity in the Gut of Mice Using Low Impedance Arrayed Microelectrodes

Smooth and elaborate gut motility is based on cellular cooperation, including smooth muscle, enteric neurons and special interstitial cells acting as pacemaker cells. Therefore, spatial characterization of electric activity in tissues containing these electric excitable cells is required for a precise understanding of gut motility. Furthermore, tools to evaluate spatial electric activity in a small area would be useful for the investigation of model animals. We thus employed a microelectrode array (MEA) system to simultaneously measure a set of 8×8 field potentials in a square area of ∼1 mm². The size of each recording electrode was 50×50 µm², however the surface area was increased by fixing platinum black particles. The impedance of microelectrode was sufficiently low to apply a high-pass filter of 0.1 Hz. Mapping of spectral power, and auto-correlation and cross-correlation parameters characterized the spatial properties of spontaneous electric activity in the ileum of wild-type (WT) and W/W^v mice, the latter serving as a model of impaired network of pacemaking interstitial cells. Namely, electric activities measured varied in both size and cooperativity in W/W^v mice, despite the small area. In the ileum of WT mice, procedures suppressing the excitability of smooth muscle and neurons altered the propagation of spontaneous electric activity, but had little change in the period of oscillations. In conclusion, MEA with low impedance electrodes enables to measure slowly oscillating electric activity, and is useful to evaluate both histological and functional changes in the spatio-temporal property of gut electric activity.     

THE EFFECT OF SPIKE ACTIVITY VERSUS SYNAPTIC ACTIVATION ON THE METABOLISM OF RIBONUCLEIC ACID IN A MOLLUSCAN GIANT NEURONE

—Previous experiments on a giant neurone (R2) from Aplysia californica have shown that a prolonged electrical stimulation of ganglionic nerves, strong enough to elicit post-synaptic spikes in the giant neurone, caused a marked increase in the uptake of labelled nucleosides into the neuronal RNA. The results described in the present paper very strongly indicate that these effects of synaptic activation were not due to the discharge of spikes in the giant neurone itself. Spikes which were directly elicited in the giant neurone by current pulses injected into the cell through an intracellular microelectrode had no significant effect on RNA labelling. Weak stimulation of ganglionic nerves, eliciting post-synaptic potentials but few spikes in the giant neurone, produced a small but significant increase of RNA labelling.     

An Assessment of the Double Sucrose-Gap Voltage Clamp Technique as Applied to Frog Atrial Muscle

The homogeneity of voltage clamp control in small bundles of frog atrial tissue under double sucrose-gap voltage clamp conditions was assessed by intracellular microelectrode potential measurements from cells in the test node region. The microelectrode potential measurements demonstrated that (1) good voltage control of the impaled cell existed in the absence of the excitatory inward currents (e.g., during small depolarizing clamp pulses of 10-15 mV), (2) voltage control of the impaled cell was lost during either the fast or slow excitatory inward currents, and (3) voltage control of the impaled cell was regained following the inward excitatory currents. Under nonvoltage clamp conditions the transgap recorded action potential had a magnitude and waveform similar to the intracellular microelectrode recorded action potentials from cells in the test node. Transgap impedance measured with a sine-wave voltage of 1,000 Hz was about 63% of that measured either by a sine-wave voltage of 10 Hz or by an action potential method used to determine the longitudinal resistance through the sucrose-gap region. The action potential data in conjunction with the impedance data indicate that the extracellular resistance (R_s) through the sucrose gap is very large with respect to the longitudinal intracellular resistance (R_i); the frequency dependence of the transgap impedance suggests that at least part of the intracellular resistance is paralleled by a capacitance. The severe loss of spatial voltage control during the excitatory inward current raises serious doubts concerning the use of the double sucrose-gap technique to voltage clamp frog atrial muscle.     

Repetitive firing triggers clustering of Kv2.1 potassium channels in Aplysia neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.     

TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm² at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.² and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero.     

Modeling error and stability of endothelial cytoskeletal membrane parameters based on modeling transendothelial impedance as resistor and capacitor in series

Transendothelial impedance across an endothelial monolayer grown on a microelectrode has previously been modeled as a repeating pattern of disks in which the electrical circuit consists of a resistor and capacitor in series. Although this numerical model breaks down barrier function into measurements of cell-cell adhesion, cell-matrix adhesion, and membrane capacitance, such solution parameters can be inaccurate without understanding model stability and error. In this study, we have evaluated modeling stability and error by using a ² evaluation and Levenberg-Marquardt nonlinear least-squares (LM-NLS) method of the real and/or imaginary data in which the experimental measurement is compared with the calculated measurement derived by the model. Modeling stability and error were dependent on current frequency and the type of experimental data modeled. Solution parameters of cell-matrix adhesion were most susceptible to modeling instability. Furthermore, the LM-NLS method displayed frequency-dependent instability of the solution parameters, regardless of whether the real or imaginary data were analyzed. However, the LM-NLS method identified stable and reproducible solution parameters between all types of experimental data when a defined frequency spectrum of the entire data set was selected on the basis of a criterion of minimizing error. The frequency bandwidth that produced stable solution parameters varied greatly among different data types. Thus a numerical model based on characterizing transendothelial impedance as a resistor and capacitor in series and as a repeating pattern of disks is not sufficient to characterize the entire frequency spectrum of experimental transendothelial impedance. cell-cell adhesion; cell-matrix adhesion; cell membrane capacitance; mathematical computation     

Laser Raman investigations of intact single muscle fibers. Protein conformations

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.     

Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing.

Transepithelial impedance of Madin-Darby canine kidney cell layers is measured by a new instrumental method, referred to as electric cell-substrate impedance sensing. In this method, cells are cultured on small evaporated gold electrodes, and the impedance is measured in the frequency range 20-50,000 Hz by a small probing current. A model for impedance analysis of epithelial cells measured by this method is developed. The model considers three different pathways for the current flowing from the electrode through the cell layer: (1) in through the basal and out through the apical membrane, (2) in through the lateral and out through the apical membrane, and (3) between the cells through the paracellular space. By comparing model calculation with experimental impedance data, several morphological and cellular parameters can be determined: (1) the resistivity of the cell layer, (2) the average distance between the basal cell surface and substratum, and (3) the capacitance of apical, basal, and lateral cell membranes. This model is used to analyze impedance changes on removal of Ca2+ from confluent Mardin-Darby canine kidney cell layers. The method shows that reduction of Ca2+ concentration causes junction resistance between cells to drop and the distance between the basal cell surface and substratum to increase.     

Estimation of surface charges in some biological membranes.

The resting membrane potential data existing in the literature for the giant axon of the squid, frog muscle and barnacle muscle have been analyzed from the standpoint of the theory of membrane potential due to Kobatake and co-workers. The average values derived for the effective charge density phi chi (where phi is a constant, 0 less than phi less than 1, and represents the fraction of counterions that are free, and chi is the stoichiometric charge density in the membrane) present on the different biomembranes existing in their normal ionic environment are 0.3, 0.325 and 0.17 M for the squid axon, frog and barnacle muscles, respectively. On the assumption that the values of phi are 0.4 and 0.2 for nerve and muscle membranes, respectively, values of 0.75, 1.62 and 0.85 M have been derived for the stoichiometric charge density (chi) present in the respective biological membranes. These correspond to 1 negative charge per 222, 103 and 195 A2 of the membrane area of the squid axon, frog and barnacle muscles, respectively.     

Dielectrophoretic manipulation of cells with spiral electrodes.

Electrokinetic responses of human breast cancer MDA-MB-231 cells were studied in suspensions of conductivities 18, 56, and 160 mS/m on a microelectrode array consisting of four parallel spiral electrode elements energized with phase-quadrature signals of frequencies between 100 Hz and 100 MHz. At low frequencies cells were levitated and transported toward or away from the center of the spiral array, whereas at high frequencies cells were trapped at electrode edges. The frequencies of transition between these characteristic cell behaviors increased with increasing suspension conductivity. Levitation heights and radial velocities were determined simultaneously for individual cells as a function of the applied field magnitude and frequency. Results were compared with theoretical predictions from generalized dielectrophoresis theory applied in conjunction with cell dielectric parameters and simulated electric field distributions corrected for electrode polarization effects. It was shown that the conventional and traveling-wave dielectrophoretic force components dominated cell levitation and radial motion, respectively. Both theoretical predictions and experimental data showed that the cell radial velocity was very sensitive to the field frequency when the in-phase component of the field-induced polarization was close to zero. Applications of spiral electrode arrays, including the isolation of cells of clinical relevance, are discussed.     

Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.