Internal Microelectrodes Research Articles

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.

The effect of the benzothiazepine diltiazem on force and Ca2+ current in isolated frog skeletal muscle fibres.

1. The action of the D-cis and L-cis isomers of the benzothiazepine derivative diltiazem on isometric force and L-type Ca2+ inward current in short muscle fibres from toe muscles of the frog (Rana esculenta and R. temporaria) was investigated under voltage clamp control with two internal microelectrodes. The experiments were performed at 10 degrees C in TEA-sulphate solution, in which the concentration of ionized Ca2+ was about 4 mM. 2. In the presence of diltiazem (D-cis, 0.25-30 microM; L-cis, 5-100 microM) normal Ca2+ currents and phasic contractures could be elicited by a depolarizing voltage step. However, after long-lasting depolarizations both the Ca2+ channel and the force controlling system (voltage sensor) remained refractory for minutes instead of seconds as under control conditions, i.e. they were 'paralysed'. L-cis diltiazem was about 20 times less effective in comparison with the D-cis isomer. 3. Speed of restoration of force and Ca2+ current was retarded by up to two orders of magnitude, depending on the applied concentration of diltiazem. 4. The steady state potential dependence of force restoration (V0.5 = -49 mV; the potential at which half of the voltage sensors are restored) and that of Ca2+ current restoration (V0.5 = -57 mV) were not altered by diltiazem. 5. The time course of the transition to paralysis, which was quite slow under control conditions, could be accelerated by diltiazem in a concentration-dependent manner. 6. Paralysis developed at the same speed when the fibre was depolarized from -90 mV to -30 mV or to +30 mV. 7. The voltage dependence of Ca2+ current inactivation (inactivation curve; conditioning pulses of 2 min duration) was shifted by about 14 mV to more negative potentials (V0.5 = -31 mV; V'0.5 = -45 mV) under the influence of 1 microM-D-cis diltiazem. It is assumed that no steady state was reached during the conditioning pulse. 8. The voltage dependence of force activation (V0.5 = -45 mV) was not altered by diltiazem. 9. Since the effects of both isomers of diltiazem could only be disclosed during the adjustment of a new steady state and since the drugs acted in a similar way on the inactivation kinetics of force and Ca2+ current, it is concluded that, in depolarized fibres, the drug facilitates the transition to a stabilized inactivated form of the voltage sensor/Ca2+ channel molecule.

The effects of dihydropyridine derivatives on force and Ca2+ current in frog skeletal muscle fibres.

1. The effects of dihydropyridine (DHP) derivatives on current through the slow Ca2+ channel and on isometric force were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage-clamp conditions with two flexible internal microelectrodes. 2. The non-chiral DHP derivative nifedipine was used mainly because it allowed control measurements after the inactivation of the drug with UV light. 3. In a TEA sulphate solution containing 4 mM-free Ca2+, nifedipine (1 microM) caused no relevant alterations in the time course of successive contractures induced by depolarizing steps to 0 mV of 3.5 min duration followed by a restoration time at -90 mV of 1.5 min. 4. When external Ca2+ was replaced by Mg2+, nifedipine caused a dose-dependent shortening of contractures. The effect reached saturation at about 50% of shortening with 1-5 microM-nifedipine. In the absence of divalent cations and with Na+ being the only metallic cation in the solution, shortening became more pronounced and maximum force decreased. 5. The application of 2 microM-nifedipine to a Ca2(+)-free, Mg2(+)-containing solution shifted the voltage dependence of force inactivation by 5-10 mV to more negative potentials. 6. Force activation was facilitated by nifedipine. In the presence of 2 microM-nifedipine in a Ca2(+)-containing solution, threshold potentials (rheobase) as negative as -75 mV were measured under microscopical observation. UV irradiation shifted the threshold potential back to the normal value of about -50 mV. 7. The slow Ca2+ inward current was blocked almost completely by approximately 5 microM-nifedipine, even when induced from negative holding potentials (-90 to -120 mV), i.e. under conditions where normal phasic contractures could still be observed. 8. Nifedipine (0.8 microM) caused a shift of the voltage dependence of current inactivation (V0.5) by 4 mV from -26 to -30 mV and at negative holding potentials (-90 mV), a reduction of maximum current by 35%. 9. The voltage dependence of current activation was not significantly altered by nifedipine (2 microM). 10. It is assumed that nifedipine binds with low affinity to the resting state of the DHP receptor (KD 0.8 microM) and with high affinity (KD 1 nM) to the inactivated and the active state (or to a precursor of this state). These assumptions could explain the relatively small shift of the inactivation curves (points 5 and 8) to more negative potentials and the facilitation of force activation (point 6).

Linear analysis ofKconduction in snail neuron from impedance determinations

Abstract The complex impedance of a soma cell membrane in an identified, isolated single neuron from Helix aspersa (snail) was measured in the frequency range 0.25 Hz to 100 Hz in order to quantify the electrical properties and kinetics of potassium conduction. Use of a synchronized Fourier-synthesized signal applied as a small perturbation during step current-source stimulation via an external suction capillary and use of an internal microelectrode to record the voltage response permitted rapid (4 sec) acquisition of a 400-point impedance function of frequency. Three types of potassium conductance were distinguishable pharmacologically in complex impedance determinations. The linear kinetic parameters of two types of K conductance, the voltage-sensitive “delayed rectifier” potassium conductance, and the Ca-activated, potassium conductance were obtained by curve fitting model admittance functions of frequency to complex admittances obtained from the reciprocal of measured steady-state impedance data. Time...

Effects of the calcium antagonist gallopamil (D600) upon excitation-contraction coupling in toe muscle fibres of the frog.

1. The effects of the Ca2+ antagonist gallopamil (D600) upon force development in short skeletal muscle fibres (m. lumbricalis digiti IV) of the frog were investigated under voltage-clamp control, using two flexible internal micro-electrodes (temperature = 6-7 degrees C). 2. In the presence of 5-100 microM-gallopamil muscle fibres developed one normal phasic contracture when they were depolarized from a holding potential of -90 to 0 mV. Subsequent depolarizations caused no mechanical response (paralysis). However, the ability to contract could be restored by hyperpolarizing the membrane to potentials between -120 and -150 mV. 3. In the absence of gallopamil, mechanical refractoriness could be fully reversed within 5-7 s by repolarizing the fibre from 0 to -120 mV. In the presence of 100 microM-gallopamil, no detectable restoration occurred within the first minute at -120 mV, and 45 to 100% of maximum force was eventually reached after 6 min of restoration. 4. The potential V at which the 'steady state' 50% of maximum force of a refractory fibre was restored shifted from -51 mV under normal conditions to -83 and -90 mV in the presence of 5 and 100 microM-gallopamil, respectively. 5. Paralysis in the presence of gallopamil and recovery from paralysis during hyperpolarization could also be observed when 2 mM-Cd2+ was applied to the external solution, i.e. when most Ca2+ channels in the T-tubular system were blocked. 6. Gallopamil shifted the threshold for activation of force to more negative potentials. Fibres developed force when they were depolarized to membrane potentials between -60 and -80 mV, whereby a fast phase of activation was followed by a slower one. Upon repolarization relaxation likewise occurred in a fast and a slow phase. 7. High concentrations of gallopamil (greater than 500 microM) caused a slowly developing contracture, independent of membrane potential (-90 or 0 mV). 8. It is proposed that gallopamil binds to a receptor at the force-controlling system in the T-tubular membrane (potential sensor) with a high affinity in the depolarized state and a lower affinity at negative potentials. Therefore association of gallopamil mainly leads to stabilization of the inactive state (paralysis) but can also stabilize the active state.

Voltage-dependent potassium channels in the amphibian lens membranes: Evidence from radiotracer and electrical conductance measurements

The electrical potential and conductance of the perfused frog lens preparation was measured using a two internal microelectrode technique. When the lens potential was depolarized by perfusing with high potassium concentration ringer the increase in conductance could not be fitted by conductance equations where P K was assumed constant. It is suggested therefore that this increase is due to a voltage-dependent component of the conductance, namely voltage-dependent potassium channels. Direct evidence for the existence of a voltage-dependent conductance is presented in the form of conductance-voltage curves which show rectification similar to squid axon. Conductance-voltage curves mapped out in high potassium Ringer show a blocking effect of potassium on the lens conductance. The blocking effect was also observed in lenses where the membrane voltage was clamped to a steady value during exposure to high potassium solutions. The increase in conductance at depolarized lens potentials is demonstrated by changes in potassium permeability as measured from the efflux of 42 K. Both the electrical conductance and 42 K efflux are shown to be sensitive to potassium channel blocking agents, namely, tetraethylammonium (TEA), caesium and rubidium ions. The 42 K efflux rate constant and lens potential were monitored simultaneously whilst depolarizing and hyperpolarizing currents were applied and the changes in 42 K efflux clearly demonstrated the voltage-dependence of the potassium permeability.

A comparison of ion concentrations, potentials and conductances of amphibian, bovine and cephalopod lenses

1. The concentrations of sodium, potassium and chloride in frog and bovine lenses showed a normal intracellular ion distribution with the sum of the internal cations approximately equal to the external sum. In the cephalopod lens, however, the sum inside was much lower than that outside.2. The membrane potentials of frog, Sepiola and bovine lenses were -63, -63 and -23 mV respectively. A comparison of the electrical data with the Nernst potentials predicted from ion concentration data indicated that sodium and chloride ions as well as potassium contributed to the membrane potential in frog and bovine. In contrast, the membrane and Nernst potentials for potassium were equal in Sepiola.3. Substituting potassium for sodium in the external medium depolarized lens potentials in all three species. Estimates of the relative permeabilities of sodium, potassium and chloride were obtained by fitting the Goldman-Hodgkin-Katz equation to the potential data.4. The potassium permeability was determined directly by (42)K efflux measurements and values of 2.99, 9.83 and 3.13 (x (-8) m sec(-1)) were obtained for frog, Sepiola and bovine lenses respectively.5. The effect of raising external potassium on the efflux rate constant was determined and there was reasonable agreement between experiment and theory (Kimizuka-Koketsu) in frog and bovine lenses, but the Sepiola data indicated that the potassium permeability decreased by a factor of 2.6 when the external potassium was raised from 10 to 120 mM-K+.6. The measured specific conductances, obtained using two internal micro-electrodes, were 7.7, 15.9 and 9.9 (Sm(-2)) for frog, cephalopod and bovine lenses respectively. These data compare with computed values (Kimizuka-Koketsu theory) of 7.5, 14.1 and 17.2 (Sm(-2)).7. The effect of increasing external potassium on the conductance was also tested and there was good agreement between experiment and theory (assuming constant permeabilities) only in the amphibian lens. However, when the cephalopod data were corrected assuming a 2.6-fold decrease in P(K) for a twelvefold increase in potassium, then there was excellent agreement between experiment and theory.8. The bovine measured conductances were much lower than the theoretical values throughout the range of external potassium concentrations and several explanations were proposed to account for the discrepancies.

Birefringence signals from surface and t‐system membranes of frog single muscle fibres.

1. When the tonicity of Ringer is increased above 2-5 times normal and a single fibre stimulated externally, the large, early birefringence signal preceding twitch tension (Baylor & Oetliker, 1975, 1977 a,b) is sufficiently reduced and delayed so as to reveal a small but distinct signal ('1st component") preceding it. For an average-sized fibre, the deltaI/I of the 1st component was (minus) 1 to 2 x 10-5. 2. The time course of the 1st component superimposed with the surface action potential simultaneously recorded by an internal micro-electrode. The polarity of the 1st component reversed with compensation. 3. From these characteristics, the 1st component is thought to arise from a small change in optical retardation of the surface membrane due to the action potential. 4. When a fibre was impaled with two micro-electrodes, retardation changes accompanying small hyperpolarizing and depolarizing current steps were detected. In some cases the polarity of the observed signal was opposite in sign to that expected for a retardation change only from the surface membrane. 5. Because the anatomical orientation of the T-system appears to be primarily transverse rather than longitudinal, these signals of opposite polarity are probably, on balance, due to retardation changes from the membranes of the T-system. 6. The possible origin of the large birefringence signal preceding contraction is discussed.

Sodium and calcium components of action potentials in the Aplysia giant neurone.

1. Action potentials resulting from direct stimulation can be recorded from the soma of the Aplysia giant neurone (located in the visceral ganglion) in sodium-free and in calcium-free external solutions. The neurones were impaled by internal micro-electrodes throughout the change of external solutions.2. Complete replacement of either sodium or calcium in the bathing medium with Tris results in only a partial reduction of spike overshoot. Simultaneous replacement of both sodium and calcium reversibly and quickly abolishes the spike.3. The sodium component of the spike in a calcium-free medium is blocked by tetrodotoxin; the drug has no effect on the calcium-dependent spike in sodium-free medium. Externally applied cobalt chloride blocks only the calcium-dependent component.4. In calcium-free media, the overshoot value varies with sodium concentration in the manner predicted for a sodium electrode. In sodium-free media, the membrane behaves like a calcium electrode.5. These results suggest that, during the normal action potential, both sodium and calcium act as carriers of the inward-directed current.

IONIC SELECTIVITY IN PERFUSED GIANT AXONS.

Information about the ionic selectivity of the sodium-carrying system of the active nerve membrane has been obtained by voltage clamp experiments on internally perfused giant axons of Loligo forbesi. These experiments were carried out in the Laboratory of the Marine Biological Association in Plymouth during the winter 1963-64. Uncleaned axons were perfused by the method of Baker, Hodgkin, and Shaw (1962). The internal microelectrode consisted of a fluid-filled capillary for measuring internal potential and a 1 cm length of platinized platinum wire for sending current. Two Ag-AgCl wires each shaped as a C were used for measuring the membrane current by the voltage it produced in the external solution. All measurements were made at approximately 0°C. The axons lasted on the average 3 to 4 hours from the time of initial perfusion. The membrane currents recorded from perfused axons with the voltage clamp technique had the same general characteristics as those recorded from intact axons (Cole, 1949; Hodgkin, Huxley, and Katz, 1952). With an isotonic solution of 305 mM KCI + sucrose inside and K-free artificial sea water outside, the membrane current associated with a small cathodal pulse consisted of a sodium inward current (Fig. 1A, record -10 my) followed by a delayed outward current. The inward component disappeared for cathodal pulses which shifted the internal membrane potential to +60 or +70 mv (Fig. 1A, record +63 my) and became outward for larger depolarizations (Fig. 1A, record +129 my). The appearance of an early outward current in the absence of internal Na strongly suggests that the internal K can partially substitute for Na in the early permeability change. The early outward current was also observed when the external Na was replaced by choline; i.e., when the internal and external solutions were both Na-free. The amplitude of the early outward current was reversibly reduced when the internal KCi was replaced by either RbCl, CsCl, or sucrose. The time course of the early outward current corresponded to the time course of the sodium permeability measured by repolarizing the membrane to the resting potential at different times after the beginning of the cathodal pulse. The steady-state inactivation of the early outward current resembled the steady-state inactivation of the sodium inward

The Electrical Resistance and Capacitance of the Membranes ofNitella translucens

SUMMARY The resistance and capacitance of the membranes of Nitella translucens have been measured by direct current and alternating current methods. Current of the order of io~7 amp. was injected into the cell by means of a conventional Ag, AgCl-3N KC1 glass microelectrode inserted into the vacuole of the cell. The change of potential across the membrane was recorded by two other internal microelectrodes which had been inserted into the cell at known distances from the current-injecting electrode. In the direct-current experiments the input current was in the form of a square pulse, while sinusoidal currents of frequency 25 cycles per second were used in the alternating current experiments. The cell was treated as a short length of coaxial cable and from the measurements the following para meters could be obtained : the space constant (A), the membrane resistance (Rm) and the membrane capacitance (Cm). The values of Rm ranged from 6-7 to 36 K ohm cm.2 (mean of 214 K ohm cm.2) and those of A ranged from 1-5 to 5-7 cm. (mean of 2-6 cm.). The capacitance value was about 1 ixF cm.-2 These results are discussed within the framework of our knowledge of these parameters for other cells, particularly plant cells. The measured electrical resistance is shown to be at least ten times less than the value estimated from the passive fluxes of the principal ions K, Na, and CI. It is suggested that this dis crepancy, which is usually attributed to non-independent movement of these ions, could be partially explained on electro-osmotic grounds. The value of the capaci tance is very close to that which is usually obtained for other cell membranes. One exceptionally low value for Nitella has been quoted in the literature. The reason for the gross error in this particular measurement is given.

Bioelectric potentials in relation to movement in amoebae

o 1. Evidence is presented to show that, in Amoeba proteus , the membrane potential (— 72 mV) and the streaming velocity (40 microns/sec) are both affected by changes in the ionic composition of the medium. 2. The membrane potential and streaming velocity are inverse functions of log external potassium chloride concentration over a wide range. Distilled water decreases the streaming velocity, although the membrane potential is raised. 3. Single penetrations in different parts of the cell and direct measurements using two internal micro-electrodes provide evidence for an electrical potential gradient in the cytoplasm along the axis of movement which approaches 1 volt/cm. The tip of a pseudopod appears to be positive with respect to the rear of the cell and negative with respect to the external medium. 4. Electrical potentials, positive or negative, applied to the rear of the cell, can be used to redirect the streaming in a chosen direction. Streaming is away from a negative electrode and towards a positive one. 5. The relationships between the membrane potential, the internal potential gradient and the motive force for streaming are discussed. A new theory is put forward for the bioelectric control of amoeboid movement.

MECHANISMS OF DIRECT AND NEURAL EXCITABILITY IN ELECTROPLAQUES OF ELECTRIC EEL

1. Current flow outward through the caudal, reactive membrane of the cell causes direct stimulation of the electroplaque. The electrical response in denervated as well as in normal preparations recorded with internal microelectrodes is first local and graded with the intensity of the stimulus. When membrane depolarization reaches about 40 mv. a propagated, all-or-nothing spike develops. 2. Measured with internal microelectrodes the resting potential is 73 mv. and the spike 126 mv. The latter lasts about 2 msec. and is propagated at approximately 1 M.P.S. 3. The latency of the response decreases nearly to zero with strong direct stimulation and the entire cell may be activated nearly synchronously. 4. Current flow inward through the caudal membrane of the cell does not excite the latter directly, but activation of the innervated cell takes place through stimulation of the nerve terminals. This causes a response which has a latency of not less than 1.0 msec. and up to 2.4 msec. 5. The activity evoked by indirect stimulation or by a neural volley includes a prefatory potential which has properties different from the local response. This is a postsynaptic potential since it also develops in the excitable membrane which produces the local response and spike. 6. On stimulation of a nerve trunk the postsynaptic potential is produced everywhere in the caudal membrane, but is largest at the outer (skin) end of the cell. The spike is initiated in this region and is propagated at a slightly higher rate than is the directly elicited response. Strong neural stimulation can excite the entire cell to simultaneous discharge. 7. The postsynaptic potential caused by neural or indirect stimulation may be elicited while the cell is absolutely refractory to direct excitation. 8. The postsynaptic potential is not depressed by anodal, or enhanced by cathodal polarization. 9. It is therefore concluded that the postsynaptic potential represents a membrane response which is not electrically excitable. Neural activation of this therefore probably involves a chemical transmitter. 10. The nature of the transmitter is discussed and it is concluded that this is not closely related to acetylcholine. 11. Paired homosynaptic excitation discloses facilitation which is not present when the conditioning stimulus is direct or through a different nerve trunk. These results may be interpreted in the light of the existence of a neurally caused chemical transmitter or alternatively as due to presynaptic potentiation. 12. The electrically excitable system of the electroplaque has two components. In the normal cell a graded reaction of the membrane develops with increasing strength of stimulation until a critical level of depolarization, which is about 40 mv. 13. At this stage a regenerative explosive reaction of the membrane takes place which produces the all-or-nothing spike and propagation. 14. During early relative refractoriness or after poisoning with some drugs (eserine, etc.) the regenerative process is lost. The membrane response then may continue as a graded process, increasing proportionally to the stimulus strength. Although this pathway is capable of producing the full membrane potential the response is not propagated. 15. Propagation returns when the cell recovers its regenerative reaction and the all-or-nothing response is elicited. 16. Excitable tissues may be classified into three categories. The axon is everywhere electrically excitable. The skeletal muscle fiber is electrically excitable everywhere except at a restricted region (the end plate) which is only neurally or chemically excitable. The electroplaque of the eel, and probably also cells of the nervous system have neurally and electrically excitable membrane components intermingled. The electroplaques of Raia and probably also of Torpedo as well as frog muscle fibers of the "slow" system have membranes which are primarily neurally and chemically excitable. Existence of a category of invertebrate muscle fibers with graded electrical excitability is also considered. 17. In the eel electroplaque and also probably in the cells of neurons, tests of the mode of neural activation carried out by direct or antidromic stimulation cannot reveal the neurally and chemically activated component. The data of such tests though they appear to prove electrical transmission are therefore inadequate for the detection and study of the chemically initiated process.

An Instrument for the Production of Microelectrodes Used in Electrophysiological Studies

The membrane potential of single muscle fibers may be measured using an internal microelectrode of tip diameter less than 0.5μ. Previously such microelectrodes could only be prepared manually and this method has certain disadvantages. The problem has been overcome through the use of an instrument for which details of design, construction, and operation are given. In principle, a 35-mm length of Pyrex tubing of outside diameter 2 mm and inside diameter 1.3 mm is heated from a surrounding electrically energized platinum coil. At the start, 100-g force is applied to the tubing in the longitudinal direction by means of a solenoid and plunger arrangement. As the tubing softens its length is extended by 5 mm whereupon the automatic application of 1700-g force draws the tubing apart thus forming two micropipettes. The dimensions and electrolytic resistance of typical microelectrodes manufactured with the aid of the instrument are presented.