Local changes of resistance in the retinal horizontal cell evoked by changes in membrane potential

A steady current (10·10^–10–6·10^–9 A) was passed by means of a bridge circuit through a recording microelectrode inserted into a horizontal cell of the turtle retina. Illumination of the retina caused an increase in the resistance of the microelectrode circuit (by 10–80 M), causing a change in the shape of the recorded response of the horizontal cell to light. The change in resistance was shown to take place, not on the cell membrane itself, but inside the cell close to the microelectrode tip. The effect described can be reproduced by passing a current through one barrel of a double-barreled microelectrode alongside the recording barrel, but the strength required for this current was greater than that passed through the recording barrel. If the membrane potential of the horizontal cell was made equal to the equilibrium potential (by means of a steady current passed through extracellular electrodes) the hyperpolarization response to light and the effect of the increase in resistance of the microelectrode circuit disappeared simultaneously. On the other hand, artificial hyperpolarization of the cell membrane caused an increase, but depolarization caused a decrease in the resistance of the microelectrode circuit. It is postulated that the observed effect is due to blocking of the microelectrode tip by an intracellular structure whose resistance varies with a change in membrane potential.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol.5, No.4, pp.432–441, July–August, 1973.     

The Spatial Variation of Membrane Potential Near a Small Source of Current in a Spherical Cell

A theoretical analysis is presented of the change in membrane potential produced by current supplied by a microelectrode inserted just under the membrane of a spherical cell. The results of the analysis are presented in tabular and graphic form for three wave forms of current: steady, step function, and sinusoidal. As expected from physical reasoning, we find that the membrane potential is nonuniform, that there is a steep rise in membrane potential near the current microelectrode, and that this rise is of particular importance when the membrane resistance is low, or the membrane potential is changing rapidly. The effect of this steep rise in potential on the interpretation of voltage measurements from spherical cells is discussed and practical suggestions for minimizing these effects are made: in particular, it is pointed out that if the current and voltage electrodes are separated by 60°, the change in membrane potential produced by application of current is close to that which would occur if there were no spatial variation of potential. We thus suggest that investigations of the electrical properties of spherical cells using two microelectrodes can best be made when the electrodes are separated by 60°.     

Intracellular gradients of electrical potential in the epithelial cells of theNecturus gallbladder

Summary When single-barrelled electrodes (5–60 M) were advanced under manual control from the mucosal side of the epithelium the mucosal membrane was on average indented by about 40 m before the microelectrode penetrated the cell. Since this dimpling was comparable with the total depth of the cell, which recovered its original shape within 0.5 sec, the steady intracellular potential was recorded only about 14 m from the basal (serosal) membrane. Fast recording of the associated change in potential revealed an abrupt drop to –26 mV at a mean rate of 84 V/sec, followed by a further slow drop to a steady value of about –50 mV at a mean rate of 0.28 V/sec. The initial level of –26 mV may be regarded as the potential difference across the mucosal membrane. This conclusion was confirmed by mounting the microelectrode on a piezoelectric probe, which delivered 3 m jabs in less than 0.5 msec. With this device in operation to prevent dimpling, the mean potential difference across the mucosal membrane was recorded as –29 mV. In all cases the potential across the basal membrane was recorded as –52 mV. Manual advance of the microelectrode tip within the cytoplasm yielded an intracellular potential gradient of 0.6 mV/m. The same potential profile and membrane potentials were demonstrated on penetrating the epithelium from the serosal side, and measurements with multibarrelled electrodes whose tips were staggered in depth gave roughly the same internal potential gradient. The resistivity of the cytoplasm was determined by a triple-barrelled microelectrode, and varied from 10 times that ofNecturus saline at the mucosal end of the cell to 4 times in the middle and 6 times at the serosal end.     

Electrical properties of rabbit corneal endothelium as determined from impedance measurements.

Alternating- and direct-current electrical characteristics of rabbit corneal endothelium were studied under varying experimental conditions. The measurements were performed by sending a 10-microA current (AC or DC) across the tissue layer. Maximal values of transendothelial potential difference and resistance were 1.3 +/- 0.1 mV and 73 +/- 6 omega . cm2, respectively. The short-circuit current was estimated from the potential and resistance values. Impedance loci were obtained for the frequency range 0.5-100 kHz. A capacitive reactance (C = 0.63 +/- 0.02 microF/cm2) was observed in the 100 Hz-100 kHz range. To relate the impedance data to the electrical parameters of the cell membranes, the voltage-divider ratio was determined by sending square pulse across the tissue and measuring voltage responses across the apical and basal membranes with an intracellular microelectrode. The intracellular potential difference was on the average -61 +/- 1 mV, and the voltage-divider ratio was found to be between 0.33 and 4. Impedance data were fit by a computer to an equivalent circuit representing a "lumped" model, and the agreement between the model and the data was satisfactory. The results are discussed in terms of both the morphological characteristics and properties of the fluid transport mechanism across the preparation.     

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.     

Intracellular microelectrode measurements in small cells evaluated with the patch clamp technique.

Microelectrode penetration of small cells leads to a sustained depolarization of the resting membrane potential due to a transmembrane shunt resistance (Rs) introduced by the microelectrode. This has led to underestimation of the resting membrane potential of various cell types. However, measurement of the fast potential transient occurring within the first few milliseconds after microelectrode penetration can provide information about pre-impalement membrane electrophysiological properties. We have analyzed an equivalent circuit of a microelectrode measurement to establish the conditions under which the peak of the impalement transients (Ep) approaches the pre-impalement resting membrane potential (Em) of small cells most closely. The simulation studies showed that this is the case when the capacitance of the microelectrode is low and the membrane capacitance of the cell high. In experiments performed to assess the reliability of Ep as a measure of Em, whole-cell patch clamp measurements were performed in the current clamp mode to monitor, free from the effects of Rs, Em in cultured human monocytes. Microelectrode impalement of such patch clamped cells and measurement of Ep made it possible to detect correlation between Ep and Em and showed that for small cells such as human monocytes Ep is on average 6 mV less negative than the resting membrane potential.     

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick’s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.     

The Voltage Dependence of the Cardiac Membrane Conductance

Solutions have been computed for the point polarization of a sheet-like membrane obeying the equations used previously (Noble, 1960, 1962) to reproduce the Purkinje fiber action potential. It was found that, in spite of the gross non-linearity of the membrane current-voltage relations, the relations between total polarizing current and displacement of membrane potential at various distances from the polarizing electrode are remarkably linear. It is therefore concluded that Johnson and Tille's (1960, 1961) results showing linear polarizing current-voltage relations obtained by passing current through the membrane from a microelectrode during the plateau of the rabbit ventricular action potential do not conflict with the Hodgkin-Huxley theory of electrical activity.     

Electrogenesis in mouse neuroblastoma cells in vitro

Intracellular microelectrode studies of passive membrane properties and action potential generation were carried out on cloned and uncloned mouse neuroblastoma cells in tissue culture. The cloned cells were studied between the eighth and tenth months and the uncloned cells between the third and fifth weeks after primary dissociation. Electrophysiologic measurements of cell membrane properties were made by passing stimulating current pulses across the cell membrane from an intracellular microelectrode and recording simultaneously from the same electrode, by means of a bridge circuit, the changes in membrane potential. The range of responses to electrical stimulation varied from passive increases in membrane potential to repetitive firing of action potentials. A 20 fold range in spike generating capability was found. Passive membrane properties (membrane potential, specific membrane resistivity, and specific membrane capacitance) were similar to those of sympathetic neurons in intact preparations. Seventy-nine percent of the cloned cell line compared to 94% of the uncloned line were capable of generating action potentials. Less than 2% of the cloned cells showed repetitive firing whereas 23% of the uncloned cells had this property. As in several types of normal neurons, the action potential mechanism was largely, although not completely, blocked by iontophoretic and bath applied tetrodotoxin.     

Unidirectional block between isolated rabbit ventricular cells coupled by a variable resistance.

We have used pairs of electrically coupled cardiac cells to investigate the dependence of successful conduction of an action potential on three components of the conduction process: (a) the amount of depolarization required to be produced in the nonstimulated cell (the "sink" for current flow) to initiate an action potential in the nonstimulated cell, (b) the intercellular resistance as the path for intercellular current flow, and (c) the ability of the stimulated cell to maintain a high membrane potential to serve as the "source" of current during the conduction process. We present data from eight pairs of simultaneously recorded rabbit ventricular cells, with the two cells of each pair physically separated from each other. We used an electronic circuit to pass currents into and out of each cell such that these currents produced the effects of any desired level of intercellular resistance. The cells of equal size (as assessed by their current threshold and their input resistance for small depolarizations) show bidirectional failure of conduction at very high values of intercellular resistance which then converts to successful bidirectional conduction at lower values of intercellular resistance. For cell pairs with asymmetrical cell sizes, there is a large range of values of intercellular resistance over which unidirectional block occurs with conduction successful from the larger cell to the smaller cell but with conduction block from the smaller cell to the larger cell. We then further show that one important component which limits the conduction process is the large early repolarization which occurs in the stimulated cell during the process of conduction, a process that we term "source loading."     

Mechanoelectrical transduction in hyaluronic acid salt solution is an entropy-driven process.

An electrical potential develops between the ends of a column of hyaluronic salt solution displaced from a resting position by gentle pressure. A previous study demonstrated that such displacement changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the salt before displacement or changing and increasing the rotation in the opposite direction, depending on the direction of the displacement. The present investigation demonstrates that the loss of bound water component across a membrane separating the solution and water is corelated with the extent of the column displacement. In addition, a return of the column to the position before displacement is correlated with a return of the water component across the membrane-but not at the same rate as the exodus. The data seem consistent with the hypothesis that the hyaluronic acid salt, when strained, adopts a less entropic configuration, releasing bound water and thus increasing the entropy of water component. This change in the distribution of entropy is reversible; i.e., Eddington's "time's arrow" is reversible with respect to the water component of the solution.     

Simultaneous Measurement of the Bioelectric Potential of the Cell Wall and the Vacuoles during the Oscillatory Response to the Nitella Cell

Simultaneous measurements of bioelectric potentials of the vacuole and cell wall in cells of Nitella mucronata were made by inserting glass microelectrodes into the vacuole and cell wall respeclively. During the oscillation of the bioelectric potential of the vacuole. induced by sudden changes of the external bathing solution or by the impalement of the cell with a microelectrode. the cell wall potential also exhibited fluctuations of variable intensities in phase and concomitant with spikes of the vacuolar potential oscillation. However, the polarity of the pulses of the cell wall potential was reverse to that of the spikes of the vacuolar potential. These results suggest that the same event is registered at both sides of the plasmalemma membrane across which these phenomena are occurring. The results also support the voltage clamp and tracer flux measurements on these cells which indicate that during the generation of single action potentials, induced by current, the plasma lemma transiently increases its permeability to Cl^? and K⁺ ions expelling them from the cell. The variable intensity of the transient hyperpolarizations of the cell wall potential is explained by the distance of the microelectrode in the cell wall from the plasmalemma.     

Electrophysiological Techniques and the Magnitudes of the Membrane Potentials and Resistances of Nitella translucens

The membrane resistance of internodal cells of Nitella translucensincreased by 50 per cent during the first 5 h after insertionof two microelectrodes into the vacuole even when precautionswere taken to eliminate external disturbances. The insertionof a third microelectrode into the cytoplasm did not affectthe resistance. In artificial pond water the final value forthe plasmalemma resistance was 112 k cm² and that for the tonoplastwas 6 k cm². The increase in the membrane potential after thefirst hour was less than 10 per cent. A recent suggestion that accurate measurements of the plasmalemmaresistance can be made with a microelectrode outside the plasmalemma,but in close contact with it, is criticized. Tests were made of the claim that leakage of current at thepoint where microelectrodes enter the cytoplasm gives rise toa local increase in current density at the tonoplast and henceleads to an overestimate of the tonoplast resistance. Valuesfor the tonoplast resistance obtained when the cytoplasmic microelectrodewas inserted through the plasmalemma were similar to those observedwhen it was pushed across the cell and inserted through thetonoplast at a point remote from the postulated current leakage.Furthermore, the tonoplast resistance stayed remarkably constantwhen the plasmalemma resistance varied in a way which wouldcause different proportions of the applied current to pass throughthe leak resistance and produce variations in the apparent tonoplastresistance. It is concluded that published values of the tonoplastresistance are not grossly inaccurate.     

Membrane capacity measurements suggest a calcium-dependent insertion of synexin into phosphatidylserine bilayers

The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffin granules and fusion of granule ghosts involves specific interactions with the lipid component of the membrane. To study the details of these interactions we measured synexin-induced changes in capacitance of phosphatidylserine bilayers formed at the tip of a patch pipet using the double-dip method. Provided calcium was present in the solution filling the pipet (10-50 mM) stable phosphatidylserine bilayers were easily formed. Addition of synexin (0.1 microgram/ml) to an external medium lacking added calcium induced no measurable changes in either bilayer resistance (10-30 G omega) or displacement current across the membrane. However, addition of calcium (0.1-2.5 mM) in the presence of synexin in the external solution caused a marked increase in the size and time constant of decay of the displacement current. From the steady-state value of the current we calculated a 5-fold decrease in resistance and from the charge displaced during the voltage-clamp pulses we calculated a 10-fold increase in membrane capacitance (from 20 to 200 fF). The size of the synexin-specific charge displacement in one direction during a pulse was always equal to the charge returning to the original configuration after the pulse. The synexin-specific transfer of charge reached saturation when the pipet potential was taken to a sufficient positive or negative value. These properties of the extra charge movement support our view that in the presence of calcium the cytosolic protein synexin penetrates into the bilayer. It is possible that these properties may be related to the mechanism by which synexin promotes membrane fusion in natural membranes.     

Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteoblastlike cells

Whole cell voltage clamp measurements using the patch technique on well-attached and well-spread cells of an osteoblastlike line (ROS 17/2.8) show the same spontaneous membrane potential activity as measurements with inserted microelectrodes. Furthermore, membrane potential measurements during the first 80 milliseconds (ms) following microelectrode penetration of the cell membrane usually show no decay. There is also good agreement between values of cell membrane resistance obtained by the microelectrode technique, the whole cell patch clamp technique, and the single channel patch clamp technique. These results indicate that our microelectrode measurements are not dominated by leak-induced artifacts, and that the spontaneous membrane potential activity is not induced by Ca2+ leakage around the microelectrode. The spontaneous membrane potential activity is eliminated in the presence of the Ca2+ ionophore A23187, also in serum-free medium, and by K+ and Ca2+ channel blockers, but it is not affected by the hyperpolarizing responses to parathyroid hormone (PTH) and dibutyryl cAMP, which persist under all of these conditions. These results support the hypothesis that the spontaneous membrane potential activity is related to repeated fluctuations of internal [Ca2+] and that such fluctuations result from a feedback loop involving Ca2+ channels or Ca2+ pumps in the cell membrane.     

The action potential in the smooth muscle of the guinea pig taenia coli and ureter studied by the double sucrose-gap method

The configuration of the electrotonic potential and the action potential observed by the double sucrose-gap method was similar to that observed with a microelectrode inserted into a cell in the center pool between the gaps. In the taenia and the ureter, the evoked spike was larger in low Na or in Na-free (sucrose substitute) solution than in normal solution. However, the plateau component in the ureter was suppressed in the absence of Na. In Ca-free solution containing Mg (3–5 mM) and Na (137 mM), the membrane potential and membrane resistance were normal, but no spike could be elicited in both the taenia and ureter. Replacement of Ca with Sr did not affect the spike in the taenia, nor the spike component of the ureter but prolonged the plateau component. The prolonged plateau disappeared on removal of Na, while repetitive spikes could still be evoked. It was concluded that the spike activity in the taenia and in the ureter of the guinea pig is due to Ca entry, that the plateau component in the ureter is due to an increase in the Na conductance of the membrane, and that both mechanisms, for the spike and for the plateau, are separately controlled by Ca bound in the membrane.     

Electrophysiology of phagocytic membranes II. Membrane potential and induction of slow hyperpolarizations in activated macrophages

The potential differences measured on the cell surface and after penetration into the cytoplasm of activated macrophages are described. Linear regressions are made of the measured potential differences as functions of the tip potential of each microelectrode. The surface potential of the macrophage is not significantly different from zero.Mouse macrophages have a transmembrane potential of ?26 mV, whereas in guinea-pig cells this value is ?18 mV. The input resistances of guinea-pig cells are higher than those of mouse macrophages. The cytoplasmic location of the electrode was characterized both by fluorescent dye injection and by electric criteria.Slow membrane hyperpolarizations are directly elicited by mechanical stimulation. Electric responses evoked by current pulses were further characterized.Our results lead to the establishment of objective criteria to validate intracellular recordings from macrophage.     

On the relation between displacement currents and activation of the sodium conductance in the squid giant axon.

The early time course of the current passing across the membrane in squid giant axons in which the ionic currents have been blocked reveals substantial asymmetries during and after the application of hyperpolarizing and depolarizing voltage-clamp pulses of identical size. Since the integral of the 'on' and 'off' current transients is zero, these currents must result from charge movements confined to the membrane and, therefore, they are nonlinear displacement currents. The steady state rearrangement of the charges as a consequence of sudden displacements of the membrane potential is consistent with a Boltzmann distribution of charges between two states characterized by different energy levels. Following changes in membrane potential the charges undergo a first order transition between these states. The relaxation time constant for the transition at a given temperature is a function of membrane potential. We propose that these displacement currents arise from a redistribution of the charges involved in the sodium gating system.     

Electrophysiology of phagocytic membranes. II. Membrane potential and induction of slow hyperpolarizations in activated macrophages.

The potential differences measured on the cell surface and after penetration into the cytoplasm of activated macrophages are described. Linear regressions are made of the measured potential differences as functions of the tip potential of each microelectrode. The surface potential of the macrophage is not significantly different from zero. Mouse macrophages have a transmembrane potential of--26 mV, whereas in guinea-pig cells this value is--18 mV. The input resistances of guinea-pig cells are higher than those of mouse macrophages. The cytoplasmic location of the electrode was characterized both by fluorescent dye injection and by electric criteria. Slow membrane hyperpolarizations are directly elicited by mechanical stimulation. Electric responses evoked by current pulses were further characterized. Our results lead to the extablishment of objective criteria to validate intracellular recordings from macrophage.     

Modeling the electrical behavior of anatomically complex neurons using a network analysis program: Passive membrane

We describe the application of a popular and widely available electrical circuit simulation program called SPICE to modeling the electrical behavior of neurons with passive membrane properties and arbitrarily complex dendritic trees. Transient responses may be calculated at any location in the cell model following current, voltage or conductance perturbations at any point. A numbering method is described for binary trees which is helpful in transforming complex dendritic structures into a coded list of short cylindrical dendritic segments suitable for input to SPICE. Individual segments are modeled as isopotential compartments comprised of a parallel resistor and capacitor, representing the transmembrane impedance, in series with one or two core resistors. Synaptic current is modeled by a current source controlled by the local membrane potential and an alpha-shaped voltage, thus simulating a conductance change in series with a driving potential. Extensively branched test cell circuits were constructed which satisfied the equivalent cylinder constraints (Rall 1959). These model neurons were perturbed by independent current sources and by synaptic currents. Responses calculated by SPICE are compared with analytical results. With appropriately chosen model parameters, extremely accurate transient calculations may be obtained. Details of the SPICE circuit elements are presented, along with illustrative examples sufficient to allow implementation of passive nerve cell models on a number of common computers. Methods for modeling excitable membrane are presented in the companion paper (Bunow et al. 1985).