Jan Gimsa

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.

Dielectrophoresis and electrorotation of Neurospora slime and murine myeloma cells

Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes.

Choosing electrodes for deep brain stimulation experiments--electrochemical considerations.

Deep brain stimulation (DBS) is a therapy of movement disorders including Parkinsons disease (PD). Commercially available electrodes for animal models of Parkinsons disease vary in geometry and material. We characterized such electrodes and found a drift in their properties within minutes and up to about 60 h after immersion in cell culture medium, both with and without a stimulation signal. Electrode properties could largely be restored by proteolytic treatment for platinum/iridium electrodes but not for stainless steel ones. Short-term drift and irreversible aging could be followed by impedance measurements. Aging was accompanied by metal corrosion and erosion of the plastic insulation. For both materials, the degradation rates depended on the current density at the electrode surfaces. Fourier analysis of the DBS pulse (60 micros, repetition rate 130 Hz) revealed harmonic frequencies spanning a band of more than three decades, with significant harmonics up to the MHz range. The band is located in a window imposed by electrode processes and capacitive cell membrane bridging at the low and high frequency ends, respectively. Even though electrode processes are reduced at higher frequencies they only vanish above 1 MHz and cannot be avoided. Therefore, the use of inert electrode materials is of special importance. The neurotoxicity of iron makes avoiding stainless steel electrodes imperative. Future developments need to avoid the use of corrosive materials and current density hot spots at the electrode surface, and to reduce low frequency components in the DBS pulses in order to diminish electrode processes.

Analytical description of the transmembrane voltage induced on arbitrarily oriented ellipsoidal and cylindrical cells.

We present an analytical equation for the transmembrane voltage (Deltaphi) induced by a homogeneous AC field on arbitrarily oriented cells of the general ellipsoidal shape. The equation generalizes the Schwan equation for spherical cells and describes the dependence of Deltaphi on field frequency, cell size and shape, membrane capacitance, conductivities of cytoplasm, membrane and external medium, the location of the membrane site under consideration, and on the orientation of the cell with respect to the field. The derivation is based on the fact that the cytoplasm and the Maxwellian equivalent body of the whole cell are both of a general ellipsoidal shape and must thus exhibit constant local fields. The constant fields allow for a relatively simple description of the potentials on the internal and external membrane sides, leading to Deltaphi. For this, the properties of cytoplasm, membrane, and external medium have been introduced into a special, finite element model. We found that Deltaphi can be unambiguously defined for non-spherical cells, provided that the membrane thickness is thin in comparison to the cell dimensions.

A Unified Resistor-Capacitor Model for Impedance, Dielectrophoresis, Electrorotation, and Induced Transmembrane Potential

Dielectric properties of suspended cells are explored by analysis of the frequency-dependent response to electric fields. Impedance (IMP) registers the electric response, and kinetic phenomena like orientation, translation, deformation, or rotation can also be analyzed. All responses can generally be described by a unified theory. This is demonstrated by an RC model for the structural polarizations of biological cells, allowing intuitive comparison of the IMP, dielectrophoresis (DP), and electrorotation (ER) methods. For derivations, cells of prismatic geometry embedded in elementary cubes formed by the external solution were assumed. All geometrical constituents of the model were described by parallel circuits of a capacitor and a resistor. The IMP of the suspension is given by a meshwork of elementary cubes. Each elementary cube was modeled by two branches describing the current flow through and around the cell. To model DP and ER, the external branch was subdivided to obtain a reference potential. Real and imaginary parts of the potential difference of the cell surface and the reference reflect the frequency behavior of DP and ER. The scheme resembles an unbalanced Wheatstone bridge, in which IMP measures the current-voltage behavior of the feed signal and DP and ER are the measuring signal. Model predictions were consistent with IMP, DP, and ER experiments on human red cells, as well as with the frequency dependence of field-induced hemolysis. The influential radius concept is proposed, which allows easy derivation of simplified equations for the characteristic properties of a spherical single-shell model on the basis of the RC model.

A comprehensive approach to electro-orientation, electrodeformation, dielectrophoresis, and electrorotation of ellipsoidal particles and biological cells.

Suspended cells may respond to AC polarization by orienting, deforming, moving or rotating. For modeling of ellipsoidal cells, a new dipole approach is proposed. Along each of the principal axis of the model, three finite elements of arbitrary but equal cross-sectional area for the interior, low conductive membrane shell and exterior are assumed. The length of the external medium elements is defined by influential radii which are related to the depolarizing factors. The model predicts the potential at the ellipsoids surface leading to the induced dipole moment. The moment obtained is identical to the Laplace approach for homogeneous ellipsoids; in the single-shell case, it is slightly different. The reason is the constant shell thickness which overcomes the confocal thickness necessary for the Laplace solution. Expressions for electro-orientation, deformation, dielectrophoresis, and electrorotation are derived. In linearly and circularly polarized fields, different orientation spectra are predicted to occur. While in linearly polarized AC fields, particles are oriented along their axis of highest polarizability, in circularly polarized fields, the axis of lowest polarizability is oriented perpendicular to the plane of field rotation. Based on this finding, a new electro-orientation method is proposed. In dielectrophoresis and electrorotation, reorientations are predicted which lead to discontinuous spectra.

A polarization model overcoming the geometric restrictions of the laplace solution for spheroidal cells: obtaining new equations for field-induced forces and transmembrane potential.

We present a new model for a variety of electric polarization effects on oblate and prolate homogeneous and single-shell spheroids. For homogeneous spheroids the model is identical to the Laplace model. For single-shell spheres of cell-like geometry the calculated difference of the induced dipole moments is in the thousandths range. To solve Laplaces equation for nonspherical single-shell objects it is necessary to assume a confocal shell, which results in different cell membrane properties in the pole and equator regions, respectively. Our alternative model addresses this drawback. It assumes that the disturbance of the external field due to polarization may project into the medium to a characteristic distance, the influential radius. This parameter is related to the axis ratio of the spheroid over the depolarizing factors and allows us to determine the geometry for a finite resistor-capacitor model. From this model the potential at the spheroids surface is obtained and, consequently, the local field inside a homogeneous spheroid is determined. In the single-shell case, this is the effective local field of an equivalent homogeneous spheroid. Finally, integration over the volume yields the frequency-dependent induced dipole moment. The resistor-capacitor approach allowed us to find simple equations for the critical and characteristic frequencies, force plateaus and peak heights of deformation, dielectrophoresis and electrorotation for homogeneous and single-shell spheroids, and a more generalized equation for the induced transmembrane potential of spheroidal cells.

Dielectric spectroscopy of human erythrocytes: investigations under the influence of nystatin

When placed in rotating electric fields red blood cells show a typical electrorotation spectrum with antifield rotation in the lower and cofield rotation in the higher frequency range. Assuming a spherical cell geometry, however, dielectrical parameters were obtained that differ from those measured by independent methods. Dielectrophoresis and, in particular, electrorotation yielded lower membrane capacitance values than expected. Introduction of an ellipsoidal model with an axis ratio of 1:2 allowed a description that proved to be consistent with dielectrophoresis and electrorotation data. For control cells an internal conductivity of 0.535 S/m, a specific membrane capacitance of 0.82 x 10(-2) F/m2, and a specific conductance of 480 S/m2 were obtained. The first characteristic frequency (frequency of fastest antifield rotation) and the related rotation speed can be measured quite quickly by means of a compensation method. Thus it was possible to follow changes of dielectric properties on individual cells after nystatin application. Ionophore-membrane interaction caused cell shrinkage in parallel to a decrease of the first characteristic frequency and rotation speed. Analysis of data revealed a decrease of the internal conductivity that is not only caused by ion loss but also, to a large extent, by a strong increase of hindrance because of shrinkage. Ionophore-induced membrane permeabilities can be calculated from volume decrease as well as from electrorotational data. In no case can these permeabilities count for the high membrane-AC conductivity that is attributed to the band-3 anion exchanging protein. The membrane-AC conductance was found not to be decreased for cells in Donnan equilibrium, which had leaked out almost completely.

Do band 3 protein conformational changes mediate shape changes of human erythrocytes

The bilayer-couple model predicts a reversible membrane crenation for an increasing ratio of external to internal monolayer area. This was comprehensively proven. However, individual erythrocytes may undergo dramatic shape changes within seconds when the suspension medium is changed. In contrast, under physiological conditions with no addition of membrane active compounds, active phospholipid translocation and passive flip-flops are comparatively slow. We propose that conformational changes of the anion-exchange protein, band 3, may rapidly alter the monolayer area ratio. Band 3 occupies about 10% of the total membrane area of human erythrocytes. Under physiological conditions, its conformers are asymmetrically distributed with about 90% of the transport sites facing the cytoplasm. This distribution is altered when external conformations are recruited by changing the transmembranous Cl- gradient, the external pH, or by the application of inhibitors. In experiments, recruitment by low ionic strength caused a rapid, temporary formation of echinocytes. This suspension effect could also be found at high ionic concentrations, when Cl- was replaced by SO4(2-). Inhibitors known to recruit the external band 3 conformation, like DIDS, SITS and flufenamic acid, are echinocytogenic. For inhibitors not recruiting a certain conformation, e.g. phenylglyoxal and niflumic acid, no shape effect was found. Since band 3 ensures a fast equilibrium of internal and external anions these ions are usually distributed according to the transmembrane potential (TMP). In the literature, a correlation of TMP and band 3 conformation, as well as a correlation of TMP and red cell shape, is described. In the proposed model, low external Cl- concentrations, inhibitors, or a negative TMP may recruit the transport sit outwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential astroglial activation in 6-hydroxydopamine models of Parkinson’s disease

In rat models of Parkinsons disease, injections of 6-hydroxydopamine (6-OHDA) into different areas of the basal ganglia result in dopaminergic neurodegeneration in the substantia nigra. The extent and time course of the dopaminergic lesions varies between the models. While the effects on neurons have been extensively studied, little is known about the effects on astrocytes. We compared astrocytic activation (i.e. increase in number and staining intensity of glial fibrillary acidic protein immunoreactive cells) at the injection site and in downstream structures of the motor loop, i.e. the globus pallidus (GP) and the subthalamic nucleus (STN) following 6-OHDA lesion of the medial forebrain bundle (MFB) or the striatum. Lesions in both regions resulted in astrocytic activation at the lesion site, but their remote effects varied. MFB injections caused astrocytic activation in the ipsi- and contralateral striatum, whereas striatal injections resulted in astrocytic activation in the GP and STN. Since 6-OHDA injections into the MFB and the striatum result in complete and partial SNc lesions, respectively, we hypothesize that communication links exist between astrocytes, or between neurons and astrocytes, along neuronal pathways that transmit activating signals in response to neuronal damage-but only if the neuronal pathways are at least partially intact.