Local development of action potentials in slow muscle fibres after complete or partial denervation.

Pyriformis muscles of Rana temporaria were completely or partially denervated by cutting the sciatic nerve or some of the small nerve branches entering the muscle. One stimulating and one to three recording microelectrodes were inserted along the fibres in order to compare the electrical activity at these points. In an early period following denervation action potentials of variable size and shape could be observed; these action potentials were often composed of two, sometimes of three or four, components. The size of individual components depended on the position of the recording microelectrode. Individual components could occasionally be triggered separately by adjusting the strength of the stimulating current pulse; propagation of these "all or none" responses was absent. In other fibres one component of the action potential could trigger another one several millimetres apart, thus indicating propagation. Conduction velocities were approximately 0.4 m/s. In partially denervated slow fibres, endplate potentials were confined to one lateral segment of the fibres, while the action potential occupied the denervated part of the membrane. The amplitudes of endplate and action potentials varied inversely with distance. Rough estimates of the length constant of the slow fibre membrane were calculated from the spatial decay of action potentials, endplate potentials and hyperpolarizing electrotonic potentials; mean values obtained were 2.5, 4.8 and 7.7 mm respectively. The results suggest that following denervation Na channels are built into discrete areas of the slow fibre membrane and that this process depends on the amount of denervation in individual fibres.     

Membrane currents in a calcium type muscle membrane under voltage clamp.

Four ionic current components were identified in the total membrane current recorded under voltage clamp conditions from the muscle membrane of the crayfish (Astacus fluviatilis). The early inward current component is dependent on the presence of Ca2+ ions, disappears in Ca2+ free solutions and is insensitive to variaton of external Na+ ions and to tetrodotoxin. The outward current consists of at least three components, an early, a late and a slow outward current. The outward currents are sensitive to TEA and their reversal potentials differ. The early potassium current may be separated in a proportion of fibres by a hump from the later potassium current. An insufficient space clamp as a cause of the hump was excluded by comparing the size of the clamped membrane area with the distribution of large membrane clefts in the fibre. The early outward current is critically dependent on the presence of Ca2+ ions and is relatively more sensitive to TEA ions and to conditioning depolarisation than the late outward current.     

Influence of the muscle fibre end geometry on the extracellular potentials

Intra- and extracellular action potentials of isolated frog muscle fibres were recorded at different distances to the end of the fibre. The first and second time derivatives of the intracellular action potentials were also recorded. The intracellular action potentials and their first and second time derivatives were almost the same regardless of the place of recording. With the decrease in the axial distance to the end the extracellular action potentials changed gradually in a complicated manner from a shape similar to the second time derivative into a shape similar to the first time derivative. Extracellular potentials, having two negative maxima, were recorded over the terminal taper part of the fibres.These alterations were simulated by a mathematical model. It was shown that the changes in the shape of the extracellular action potentials around the end of the fibres were mainly due to the existence of the fibre end though a better correspondence of the experimentally recorded and the calculated extracellular action potentials was obtained when the morphology of the fibre end was taken into consideration.     

Inactivation of calcium current in the somatic membrane of snail neurons

The decline of calcium inward currents evoked by a long-lasting membrane depolarization was studied on isolated snail neurons internally perfused with a K+-free solution. Two exponential components superimposed on a steady inward current could be distinguished, a slow decline with a time constant of several hundreds of milliseconds, observed at all the testing potentials used, and a fast one with a time constant of several dozens of milliseconds, which appeared at depolarizations to about -10 mV and above. When the calcium current was blocked by extracellular Cd2+ or verapamil, an outward current could be recorded at the same depolarizations. Subtraction of the latter current from the total current, recorded prior to the blockage, largely reduced the fast component of the decline of the total current. An increase in pHi from 7.3 to 8.1 led to the elimination of both the outward current and the fast component of the calcium current decline. The slow component remained practically unchanged, with its rate depending upon the current amplitude. It was slowed following intracellular administration of EDTA, and after equimolar substitution of Ba2+ for Ca2+. It is concluded that the fast component of the calcium inward current decline is mainly due to the superposition of the outward current produced by low selective channels. Only the slow component represents an actual decline of the inward current through calcium channels; it is due to ion accumulation at the inner surface of the cell membrane.     

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

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

Extracellular action potentials of skeletal muscle fibre affected by 4-aminopyridine: a model study

A new specially designed analytical function approximating the intracellular action potentials (ICAPs) for calculation of the extracellular potentials (ECAPs) at various radial and axial distances from the active fibre is proposed. 4-Aminopyridine (4-AP) was used to obtain ICAPs with a prolonged repolarization phase in order to investigate the influence of changes in ICAP shape on the ECAPs. From the experimentally recorded ICAPs before and after treatment of frog skeletal muscle fibres with 4-AP, approximated by the new function, the ECAPs were calculated applying the linesource model in a finite fibre. Using this function allowed calculation of the ECAPs at distances not accessible for the experimental recordings. The total ionic current (I _i) during the action potential was calculated using the cable equation. Our results showed that the ratio of the first positive to the negative phases of the ECAPs of treated fibres increased at large radial distances (3000 m and more) and the terminal positive phase was asymmetric with an abrupt initial deflection followed by a slow inverse deflection. The calculated ECAPs at various axial distances from the fibre end (cylindrical and conical part) and at radial distances from the fibre membrane ranging from 0 to 5000 m, corresponded in shape to the experimentally recorded potentials of untreated and 4-AP-treated muscle fibres.     

Calcium current in embryonic Xenopus muscle cells in culture

We have investigated the appearance of calcium current in Xenopus muscle cells in 1- to 6-day-old cultures. Whole cell currents were recorded using a patch-clamp amplifier with sodium and potassium replaced with tetraethylammonium and cesium, respectively, and BaCl2 used in place of CaCl2. When the muscle membrane was depolarized above -30 mV, a slow inward current was activated, the current reached a peak amplitude near 0 mV, and an outward current became apparent above +10 mV. This slow current was enhanced by adding barium or Bay K 8644 to the extracellular recording solution and was blocked by the addition of cobalt, cadmium, or the dihydropyridines nifedipine or (+)PN 200-110. Taken together these results indicate the presence of an inward calcium current mediated through L-type channels. Thirty-one percent of the cells examined on the first day in culture showed no discernible slow inward current; however, as the age of the culture increased, all cells showed slow inward current and there was an increase in the amplitude of the current. A small proportion of the muscle cells (5 out of 34) also showed a fast activating and inactivating inward current. This current, which activated at more hyperpolarized potentials (-40 mV) was only present when 5 mM ATP was included in the internal recording solution. It also appeared to be mediated through a calcium channel but not a dihydropyridine, sensitive channel.     

Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH.

We have used three-microelectrode voltage clamp in conjunction with the ammonium prepulse method to investigate the effects of lowered intracellular pH (pHi) on resting potassium currents of frog skeletal muscle fibres. Potassium currents were recorded in 40 mM K+, Cl(-)-free solution in response either to voltage steps or ramps. An ammonium prepulse (2 h) reduced pHi to 6.45 from a control value of 7.19. The intracellular ATP concentration, measured with high-pressure liquid chromatography (HPLC), was unchanged by this procedure. Mean outward potassium currents were larger in low pHi than in control fibres, being about twice as large at +40 mV, whereas mean inward currents were very similar in control and low-pHi fibres. The sulphonylurea glibenclamide blocked single KATP channels in excised patches with a Kd of 3 microM. In intact fibres 50 microM glibenclamide had no effect on K+ currents in controls but reduced currents in low-pHi fibres. In the presence of glibenclamide, K+ currents in low-pHi fibres were not significantly different from those in control fibres. We suggest that reduced pHi in intact skeletal muscle fibres opens ATP-dependent potassium channels (KATP channels), as has been shown to occur in excised patches of membrane.     

Spectral and time domain characteristics of single muscle fibre action potentials during continuous activity extracted from model considerations

A model of the muscle fibre extracellular action potentials (ECAPs) calculation using experimentally recorded intracellular action potentials (ICAPs) has been applied to investigate the effect of repetitive stimulation on the electrical activity of isolated frog muscle fibres. The ECAPs were calculated both at small (0.01 mm) and at large (5 mm) radial distances to the fibre axis, and their relationship with the original ICAP parameters has been inferred. Fourier transformation of the calculated ECAPs in order to obtain the spectral characteristics and to trace out their behaviour during continuous fibre activity was performed. Stimulation frequency dependence on the ECAP time characteristics and on the shift of the maximum spectral density towards low frequencies at small and large radial distance were observed. The spectral density peak frequency is propagation velocity (PV)-dependent. The advantage of the presented method over the available experimental extracellular recording techniques from isolated muscle fibers is the possibility to show the effect of continuous muscle fibre activity on the parameters of the ECAPs and their spectral characteristics at large radial distance, which is not experimentally accessible. Our results are in agreement with those experimentally obtained. The results from the model prove the role of changes in PV of excitation along the muscle fibres (representing the last link in the complex organized motor system) in the development of fatigue. Received: 24 July 1997 / Accepted in revised form: 2 July 1998     

Calcium channels in crayfish muscle fibre fragments studied by means of the Vaseline gap technique

Calcium currents in crayfish muscle fibres were studied by means of the vaseline gap voltage clamp technique. Overlapping potassium currents were fully suppressed using fibre fragments equilibrated in K+-free intracellular solution. The design of the recording chamber tailored to crayfish muscle fibres is described in detail. Ca currents recorded has a two-component time course. The transient (ICa, T) component (peaking in about 10 ms) attained, on average, maximal overall density of 26.4 microA/cm2 at depolarization to -4.6 mV from a holding potential of -80 mV. The steady (ICa, S) component attained 16.7 microA/cm2 (evaluated at the end of a 70 ms pulse) at +13.8 mV. The average overall surface area of the clamped membrane surface (including invaginated parts) was about 0.07 cm2. The ICa, S component could be separated from ICa, T using short inactivating prepulses. Voltage and time dependence of the transient component inactivation, as well as its recovery from inactivation, were in agreement with a Ca-dependent mechanism. Independent behaviour of the two Ca current components and differences in their properties support the hypothesis concerning the existence of two populations of Ca channels in the surface membrane of the crayfish muscle.     

Funnel web spider venom produces spontaneous action potentials in nerve

Venom from the lethal Australian spider, $\text{Atrax robustus}$, causes fasciculation of muscles $\text{in vivo}$ and in isolated diaphragms in mice. Spontaneous end-plate potentials were recorded in muscle fibres exposed to the venom and associated spontaneous electrical activity could also be recorded from the phrenic nerve. It was proposed that the venom produces muscle fasciculation by causing abnormal, spontaneous, repetitive firing of motor nerves. The mechanism of this action was investigated in aplysia neurones. The venom produced abnormal, spontaneous, repetitive inward currents in voltage clamped neurones and changed the current-voltage characteristics of the surface membrane. It is suggested that the basic mode of action of Funnel-web venom is to change the electrical field in nerve membrane.     

Calcium-dependent sodium current in the marine ciliateEuplotes vannus

Summary Ca and Na inward currents were recorded upon depolarizations inEuplotes after the blockage of K outward currents with intracellular Cs ions. The Na current was analyzed under voltage clamp and had the following properties: it activated to a maximum within 150 msec and partly inactivated during sustained voltage steps. It had a positive equilibrium potential between 25 and 30 mV and could be carried by Na or Li ions but not by K, choline or Tris ions. The current revealed a prominent associated inward tail current which deactivated with a single-exponential time constant of 118 msec. Both the current and its tail were strongly reduced after reduction of the extracellular Na concentration. Externally applied K channel blocker tetraethylammonium chloride did not block the current. Either EGTA injection into the cell or nonlethal deciliation with ethanol eliminated the current and its tail. These results indicate the existence of a Na conductance within the membrane ofEuplotes which is activated by the intracellular level of free Ca²⁺.     

Calcium currents of frog and insect skeletal muscle fibres measured during voltage clamp

Both vertebrate and invertebrate skeletal muscle fibres have Ca2+ permeability mechanisms which are turned on by depolarization of the surface membrane. In frog muscle, Ca currents are extremely slow and will be scarcely activated during the action potential that normally elicits a twitch. This Ca permeability cannot therefore play any substantial, direct role in excitation--contraction coupling. In insect (Carausius morosus) muscle, Ca currents activate within milliseconds of depolarization, even at low temperature, and may well play at least a triggering role in excitation--contraction coupling. These Ca currents show saturation with increasing [Ca]0, while the instantaneous current--voltage relation rectifies inwards, as expected from a very low [Ca]i. The Ca channel is permeable to Sr2+ and Ba2+. Inactivation of Ca currents under a maintained depolarization depends on Ca2+ carrying inward current, however, rather than on the depolarization itself.     

Voltage clamp analysis of embryonic heart cell aggregates

The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.     

Adrenergic control of cardian pacemaker currents.

Pacemaker activity in atrial muscle and in Purkinje fibres is generated by a time-dependent decay of potassium current that allows the membrane to be depolarized to the threshold for action potential initiation. The kinetics of the pacemaker potassium currents in these two parts of the heart are sufficiently different to indicate that they correspond to different membrane structures. This conclusion is strengthened by the discovery that the mechanisms of acceleration produced by adrenaline are also quite different. In Purkinje fibres, the activation threshold for the potassium current is shifted in a depolarizing direction with no change in maximum amplitude. This voltage shift is adequate by itself to explain the acceleration. In atrial fibres the pacemaker potassium current is increased in amplitude with no shift in threshold. By itself, this action of adrenaline would slow pacemaker activity and the acceleration in this case is dependent on a large increase in the current attributable to calcium ions. The roles of cyclic 3',5'-AMP and of intracellular calcium ions in mediating the pacemaker actions of adrenaline will also be discussed.     

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue. Received: 5 May 1998 / Revised version: 27 October 1998 / Accepted: 19 January 1999     

Effect of ryanodine on the electrical and mechanical activity of frog atrial fibers

The effect of ryanodine on the action potential, slow inward current and mechanical activities of frog atrial fibres was studied by means of the double sucrose gap technique. Ryanodine was shown to reduce the amplitude of the slow inward current, to cause an intracellular Ca accumulation and to decrease the tonic component of the tension.     

Fibre conduction velocity and fibre composition in human vastus lateralis

The relationship between muscle fibre composition and fibre conduction velocity was investigated in 19 male track athletes, 12 sprinters and 7 distance runners, aged 20-24 years, using needle biopsy samples from vastus lateralis. Cross sectional areas of the fast twitch (FT) and slow twitch (ST) fibres were determined by histochemical analysis. The percentage of FT fibre areas ranged from 22.6 to 93.6%. Sprinters had a higher percentage of FT fibres than distance runners. Muscle fibre conduction velocity was measured with a surface electrode array placed along the muscle fibres, and calculated from the time delay between 2 myoelectric signals recorded during a maximal voluntary contraction. The conduction velocity ranged from 4.13 to 5.20 m.s-1. A linear correlation between conduction velocity and the relative area of FT fibres was statistically significant (r = 0.84, p less than 0.01). This correlation indicates that muscle fibre composition can be estimated from muscle fibre conduction velocity measured noninvasively with surface electrodes.     

Changes in the muscle fibre extracellular action potentials in long-lasting (fatiguing) activity

The dependence of extracellular action potentials (ECAPs) of single frog muscle fibres on intracellular action potentials (ICAPs) was studied during long-lasting (fatiguing) activity. The conduction velocity, peak-to-peak amplitude and amplitudes of the separate phases of the first and second ICAP time derivatives decreased during long-lasting activity. The phases of the first and second ICAP space derivatives also decreased in amplitude and lengthened. ECAPs near the membrane were similar in shape and proportional in amplitude to (formula; see text) when recording at a distance from both the end of the fibre and the point of stimulation. At long radial distances, the amplitudes of the separate ECAP phases depended on the amplitude and length of the corresponding phases of (formula; see text). Thus the decrease in ECAP amplitude during long-lasting activity at long radial distances was less than at points close to the muscle fibre membrane. The consequences of these findings for the changes in electromyograms recorded by needle or superficial electrodes during long-lasting (fatiguing) activity are discussed.     

Selectivity of sodium channels in denervated tonic muscle fibre membrane of the frog

Ionic selectivity of sodium channels was examined under voltage clamp conditions in normal and denervated twitch fibres and denervated tonic fibres isolated from m. ileofibularis of the frog (R. temporaria). Membrane currents were recorded by means of the Hille-Campbell vaseline-gap voltage clamp method from muscle fibre segments exposed to a potassium-free artificial internal solution. Permeability ratio (PS/PNa) were determined from changes in the reversal potential after replacing all Na ions in the solution bathing the voltage clamped external membrane area with sodium substituting ions (S). The permeability sequence was: Na+ greater than Li+ greater than NH4+ greater than K+. No inward currents were observed for Ca2+. The permeability ratios were as follows. Denervated tonic fibres: 1:0.88:0.23:0.012; control twitch fibres: 1:0.94:0.22:0.076; denervated twitch fibres: 1:0.91:0.14:0.082. The permeability to Li+ ions deviates from independence to a greater extent in tonic than in phasic fibres. Our results are consistent with the Hille model of sodium channel selectivity, and they support the hypothesis that sodium channels formed in denervated tonic muscle fibres of the frog are of the same genetic origin as Na channels expressed under physiological conditions.