Impedance of a Small Electrically Isolated Area of the Muscle Cell Surface

A method has been developed permitting measurement of membrane impedance and current, as a function of transmembrane potential, at small, electrically isolated regions of the muscle cell surface without microelectrode impalement. The frequency dependence of the muscle cell membrane capacity found earlier by other methods has been confirmed. The average capacity is 3.5 µf/cm² with a phase angle of 71° at 5 kilocycles. The internal phase angle of the complex impedance plot of whole muscle probably does not result from a distribution of fiber diameters and membrane capacities, since it also appears in the present experiments where measurements are confined to a small region of a single fiber.     

The Interpretation of Current-Voltage Relations Recorded from a Spherical Cell with a Single Microelectrode

An analysis is presented of the displacement of potential recorded when one microelectrode is used both to apply current to and record potential from a spherical cell. There are three significant components of the displacement in potential: a component produced inside the microelectrode, a time-independent component representing the spatially nonuniform flow of current in the immediate vicinity of the microelectrode, and a time-dependent spatially uniform component representing the average potential across the cell membrane. The second component describes changes in the potential across the cell membrane as well as potential drops in the interior of the cell, the importance of each factor being dependent on the location of the electrode. Simple expressions, derived by a theoretical treatment, are given for each component of potential. The implications for the interpretation of experimental results determined with the "single-electrode bridge" technique are discussed and an optimal balancing procedure is suggested.     

Effect of Temperature on the Potential and Current Thresholds of Axon Membrane

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 µa at 267deg;C to 1 µa at 7.5°C. Threshold potential values ranged from 50 mv at 26°C to 6 mv at 7.5°C. The mean Q₁₀ of threshold current was 2.3 (SD = 0.2), while the Q₁₀ for threshold potentials was 2.0 (SD = 0.1).     

The capacitance of skeletal muscle fibers in solutions of low ionic strength

The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 µF/cm² in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 µF/cm². It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 µF/cm² which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential—like mechanism in the tubular membrane.     

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential

The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (± standard deviation) for unplasmolyzed tobacco cells was −52 ± 12 millivolts, for cells in 0.3 molar mannitol, −50 ± 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, −49 ± 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was −49 ± 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells.     

Reversal potential for monosynaptic EPSP in frog motoneurons

Experiments were conducted on brain isolated from the frogRana ridibunda using a current chop technique of transmembrane polarization and discrete measurement of membrane potential by a single microelectrode during intervals between waves of current. It was found that the current-voltage relationship of the motorneuron is non-linear; i.e., membrane resistance decreases considerably in step with increased depolarizing current. After the initial reduction, membrane resistance began to climb back when a more protracted current lasting 1–2 min was applied; consequently membrane potential level shifted towards more positive values of +50 mV and above at current levels of 40–60 nA. It then became possible to bring about complete reversal of monosynaptic EPSP produced in the lumbar motoneurons by stimulation of the brainstem reticular formation or by microelectrode stimulation of the ventrolateral tract descending fibers and to measure reversal potential of these EPSP directly, without resorting to computing or extrapolation. Measurements varied mainly between 0 and –10 mV.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 534–542, July–August, 1986.     

The Structure of Mytilus Smooth Muscle and the Electrical Constants of the Resting Muscle

The individual muscle fibers of the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. are uninucleate, 1.2–1.8 mm in length, 5 µm in diameter, and organized into bundles 100–200 µm in diameter, surrounded by connective tissue. Some bundles run the length of the whole muscle. Adjacent muscle cell membranes are interconnected by nexuses at frequent intervals. Specialized attachments exist between muscle fibers and connective tissue. Electrical constants of the resting muscle membrane were measured with intracellular recording electrodes and both extracellular and intracellular current-passing electrodes. With an intracellular current-passing electrode, the time constant τ, was 4.3 ± 1.5 ms. With current delivered via an extracellular electrode τ was 68.3 ± 15 ms. The space constant, λ, was 1.8 mm ± 0.4. The membrane input resistance, R_eff, ranged from 23 to 51 MΩ. The observations that values of τ depend on the method of passing current, and that the value of λ is large relative to fiber length and diameter are considered evidence that the individual muscle fibers are electrically interconnected within bundles in a three-dimensional network. Estimations are made of the membrane resistance, R_m, to compare the values to fast and slow striated muscle fibers and mammalian smooth muscles. The implications of this study in reinterpreting previous mechanical and electrical studies are discussed.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

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.     

Inhibitory Synapses on Pacemaker Neurons in the Heart Ganglion of a Stomatopod, Squilla oratoria

The pacemaker neurons of the Squilla heart ganglion are innervated from the CNS through three pairs of extrinsic nerves. One of them, the α-nerve, is inhibitory to the heart beat. The effect of α-nerve stimulation on the pacemaker potential was examined with intracellular electrodes. Without extrinsic nerve stimulation the membrane potential of the pacemaker cell fluctuated spontaneously. On application of a tetanic train of stimuli to the α-nerve the membrane potential was shifted and fixed to a steady level, which with K₂SO₄-filled electrodes was near the peak of hyperpolarization after a spontaneous burst, but was less negative with KCl-filled electrodes. The shift of the membrane potential was due to the summated IPSP's. By changing the level of the membrane potential with injection of the polarizing current the IPSP could be reversed in sign, and the size of the IPSP was linearly correlated with the membrane potential level. During inhibition the membrane conductance increased. The increase depended on divalent cation concentrations in the outside medium. In Ca-rich saline the IPSP was greatly enhanced. In Mg-rich saline it was suppressed. The amplitude of antidromic spikes was reduced during inhibition especially when the spike frequency was high.     

Adaptation in the Visual Cortex: Influence of Membrane Trajectory and Neuronal Firing Pattern on Slow Afterpotentials

The input/output relationship in primary visual cortex neurons is influenced by the history of the preceding activity. To understand the impact that membrane potential trajectory and firing pattern has on the activation of slow conductances in cortical neurons we compared the afterpotentials that followed responses to different stimuli evoking similar numbers of action potentials. In particular, we compared afterpotentials following the intracellular injection of either square or sinusoidal currents lasting 20 seconds. Both stimuli were intracellular surrogates of different neuronal responses to prolonged visual stimulation. Recordings from 99 neurons in slices of visual cortex revealed that for stimuli evoking an equivalent number of spikes, sinusoidal current injection activated a slow afterhyperpolarization of significantly larger amplitude (8.5±3.3 mV) and duration (33±17 s) than that evoked by a square pulse (6.4±3.7 mV, 28±17 s; p<0.05). Spike frequency adaptation had a faster time course and was larger during plateau (square pulse) than during intermittent (sinusoidal) depolarizations. Similar results were obtained in 17 neurons intracellularly recorded from the visual cortex in vivo. The differences in the afterpotentials evoked with both protocols were abolished by removing calcium from the extracellular medium or by application of the L-type calcium channel blocker nifedipine, suggesting that the activation of a calcium-dependent current is at the base of this afterpotential difference. These findings suggest that not only the spikes, but the membrane potential values and firing patterns evoked by a particular stimulation protocol determine the responses to any subsequent incoming input in a time window that spans for tens of seconds to even minutes.     

Excitation-contraction coupling in a barnacle muscle fiber as examined with voltage clamp technique

Relations between the membrane potential and the tension associated with changes in membrane potential were analyzed in barnacle giant muscle fibers by using voltage clamp techniques. With a step change in membrane potential the tension reaches its final level with a time course which is expressed by the difference of two exponential functions. The time constants τ₁ (0.2–0.4 sec at 23°C) and τ₂ (0.07–0.12 sec at 23°C) are independent of the new membrane potential at least for a relatively small membrane potential change while the final level of tension is a function of the potential. Decreasing the temperature increases both τ₁ and τ₂ (Q₁₀ = -2 to -3) and the increase of the tonicity of the external medium increases τ₁ but not τ₂. The final level of tension is related by an S-shaped curve to the membrane potential. The slope of the final tension-membrane potential curve increases with increasing external Ca concentration and is reduced when a small amount of transition metal ions is added to the medium. This suggests that the influx of Ca ions through the membrane is an important factor in the development of tension.     

Current- and Voltage-Clamped Studies on Myxicola Giant Axons : Effect of tetrodotoxin

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm² and 1.22 KΩ cm ² respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.     

Electric current precedes emergence of a lateral root in higher plants

Stable electrochemical patterns appear spontaneously around roots of higher plants and are closely related to growth. An electric potential pattern accompanied by lateral root emergence was measured along the surface of the primary root of adzuki bean (Phaseolus angularis) over 21 h using a microelectrode manipulated by a newly developed apparatus. The electric potential became lower at the point where a lateral root emerged. This change preceded the emergence of the lateral root by about 10 h. A theory is presented for calculating two-dimensional patterns of electric potential and electric current density around the primary root (and a lateral root) using only data on the one-dimensional electric potential measured near the surface of the primary root. The development of the lateral root inside the primary root is associated with the influx of electric current of about 0.7 μA·cm⁻² at the surface.     

Effects of Temperature on the Generator and Action Potentials of a Sense Organ

Charge transfer through the receptor membrane of the nonmyelinated ending of Pacinian corpuscles is markedly affected by temperature. The rate of rise and the amplitude of the generator potential in response to a constant mechanical stimulus increase with temperature coefficients of 2.5 and 2.0 respectively. The duration of the falling phase, presumably a purely passive component, and the rise time of the generator potential are but little affected by temperature. The following interpretation is offered: Mechanical stimulation causes the conductance of the receptor membrane to increase and ions to flow along their electrochemical gradients. An energy barrier of about 16,000 cal/mole limits the conductance change. The latter increases, thus, steeply with temperature, causing both the rate of rise and the intensity of the generator current to increase. The membrane of the adjacent Ranvier node behaves in a distinctly different manner. The amplitude of the nodal action potential is little changed over a wide range of temperature, while the durations of its rising and falling phases increase markedly. The electrical threshold of the nodal membrane is rather constant between 40 and 12°C. Below 12°C the threshold rises, and the mechanically elicited generator current fails to meet the threshold requirements of the first node. Cold block of nerve impulse initiation then ensues, although the receptor membrane still continues to produce generator potentials in response to mechanical stimulation.     

Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.     

Spectral Studies of a Chlorophyll Pigment with Fluorescence Maximum at 698 mμ

A chlorophyll type pigment (F698) fluorescing maximally at 698 mμ at 77°K has been observed in preparations of chlorophyll. This fluorescence is quenched by small amounts of naturally occurring materials, including plastoquinone and the ubiquinones, and by nitrobenzene, probably by formation of a nonfluorescent complex. Fluorescence quenching does not occur in the presence of carotenes, xanthophylls, or reduced plastoquinone and ubiquinone. The fluorescence is sharply temperature dependent, with a steep rise in intensity occurring at 165°K. At 77°K the fluorescence yield is between 0.8 and 1.0. The red absorption maximum of the pigment is at 675 mμ at room temperature and at 688 mμ at 77°K. In vivo, a low temperature emission is also observed at 698 mμ, and this fluorescence is quenched by nitrobenzene. It is proposed that the pigment found in vitro is also the one responsible for emission at 698 mμ in vivo. A reaction of F698 with plastoquinone is suggested as the primary photochemical step in system II of photosynthesis.     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.     

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.     

Internal citrate ions reduce the membrane potential for contraction threshold in mammalian skeletal muscle fibers.

An effect of internal citrate ions on excitation-contraction coupling in skeletal muscle is described. The threshold for contraction was measured in rat extensor digitorum longus, (EDL), and soleus muscle fibers using a two microelectrode voltage clamp technique with either KCl-filled or K3 citrate-filled current electrodes. Contraction thresholds were stable for many minutes with KCl current electrodes. In contrast, thresholds fell progressively towards the resting membrane potential, by as much as -15 mV over a period of 10 to 20 min of voltage-clamp with citrate current electrodes. In addition, prepulse inhibition was suppressed, subthreshold activation enhanced and steady-state inactivation shifted to more negative potentials. Fibers recovered slowly from these effects when the citrate electrode was withdrawn and replaced with a KCl electrode. The changes in contraction threshold suggest that citrate ions act on the muscle activation system at an intracellular site, since the citrate permeability of the surface membrane is probably very low. An internal citrate concentration of 5 mM was calculated to result from citrate diffusion out of the microelectrode into the recording area for 20 min. 5 mM citrate added to an artificial cell lowered the free calcium concentration from 240 to 31 microM. It is suggested that citrate modifies excitation-contraction coupling either by acting upon an anion-dependent step in activation or by reducing the free calcium and/or free magnesium concentration in the myoplasm.