Regenerating mammalian nerve fibres: changes in action potential waveform and firing characteristics following blockage of potassium conductance

Extracellular application of potassium channel blocking agents is known to increase the amplitude and duration of the compound action potential in non-myelinated and demyelinated axons, but not in mature mammalian myelinated fibres. In the present study we used intra-axonal and whole nerve recording techniques to study the effects of the potassium channel blocking agent 4-aminopyridine (4-AP) on regenerating rat nerve fibres. Our results indicate that early regenerating (premyelinated) axons show considerable broadening of the action potential after 4-AP application and late regenerating (myelinated) axons give rise to burst activity following a single stimulus after 4-AP application. 4-AP did not affect spike waveform or firing properties of normal mature sciatic nerve fibres. These results demonstrate the importance of potassium conductance in stabilizing firing properties of myelinated regenerating axons.     

Differences between mammalian ventral and dorsal spinal roots in response to blockade of potassium channels during maturation

Differences in potassium channel organization between motor and sensory fibres have been described in amphibians but have not previously been examined in mammals. In the present investigation, we studied whole nerve and single axon responses following pharmacological blockade of potassium conductance in rat ventral and dorsal spinal roots during maturation. Our results indicate a differential sensitivity in maturing mammalian motor and sensory fibres which is most apparent in younger roots. Specifically, application of 4-aminopyridine (4-AP) results in a broadening of the compound action potential in ventral roots which is associated with a delayed repolarization of the individual action potential of single fibres. In contrast, blockade of potassium channels in young dorsal roots results in a late negativity in the compound response which is correlated with multispike bursting activity recorded from single sensory fibres. The effects of 4-AP on ventral root fibres diminish earlier in the course of maturation than do the effects of 4-AP in dorsal root fibres. These results demonstrate developmental differences in the functional organization of potassium channels in mammalian motor and sensory axons which may have implications for differences in coding properties between these two classes of axons.     

The role of slow potassium current in phenomena of voltage-dependent action of 4-aminopyridine in amphibian myelinated nerve fibres

The mechanism underlying the voltage-dependent action of 4-aminopyridine (4-AP) is investigated in experiments on amphibian myelinated nerve fibres (Rana ridibunda Pallas) by way of extracellular recording of electrical activity and using activators of potassium current (potassium-free solution and nitric oxide NO) and inhibitors of sodium current (tetrodotoxin). Measurement of action potential (AP) areas was used to evaluate the extent of general membrane depolarization during the activity of nerve fibres. Tetrodotoxin-induced decrease in general membrane depolarization (when the action potential amplitude was reduced by less than 20%) leads to an increase in the duration of depolarizing after-potential (DAP). This supports the dependence of time course of DAP in the presence of 4-AP on ratio of fast and slow potassium channels. In the absence of 4-AP, potassium-free solution and NO increase the potassium current through fast potassium channels (decreasing AP duration, reducing DAP and sometimes producing fast hyperpolarizing after-potential (HAP) after shortened AP), and in the presence of 4-AP these activators increase potassium current through unblocked slow potassium channels (making the development of slow HAP induced by 4-AP more rapid). The increase of slow HAP induced by 4-AP under the influence of potassium-free solution with NO supports the idea that slow HAP is due to activation of slow potassium channels and argues against the notion of removal of block of fast potassium channels. All analyzed phenomena of voltage-dependent action of 4-AP in amphibian myelinated nerve fibers can be accounted for by the activation of slow potassium current produced by membrane depolarization and a decrease of the amount of fast potassium channels involved in the membrane repolarization.     

The role of fast and slow potassium currents in electrical activity of amphibian myelinated nerve fibres

The paper reviews the information about the role of fast and slow potassium currents in electrical activity of amphibian myelinated nerve fibres. It demonstrates the importance of discovering of fast and slow potassium currents and their following pharmacological separation (by potassium channels blockers 4-aminopyridine and tetraethylammonium) in investigation of mechanisms of biological potentials generation. The information about the existence of fast and slow potassium channels in the nerve membrane and about the properties of 4-aminopyridine and tetraethylammonium action served as a base for determination the nature of biological potentials and discovering the mechanism of potential-dependent action of 4-aminopyridine that for tens of years suffered from the lack of adequate explanation.     

Conduction along myelinated and demyelinated nerve fibres with a reorganized axonal membrane during the recovery cycle: model investigations

The changes in the excitability of the reorganized axonal membrane in myelinated and demyelinated nerve fibres as well as the causes conditioning such changes have been investigated by paired stimulation during the first 30 ms of the recovery cycle. The variations of the action potential parameters (amplitude and velocity) are traced also. The simulation of the conduction along the normal fiber is based on the Frankenhaeuser and Huxley (1964) and Goldman and Albus (1968) equations, while the demyelination is considered to be an elongation of the nodes of Ranvier. The axonal membrane reorganization is achieved by means of potassium channel blocking and increase of the sodium-channel permeability. It is shown that potassium channels block decreases membrane excitability for the myelinated and demyelinated fibres in the cases of initial and paired stimulation. With increasing sodium-channel permeability on the background of the blocked potassium channels, the membrane excitability is increased. For the fibres with a reorganized membrane, a supernormality of the membrane excitability is obtained, the latter remaining unrecovered during the 30 ms cycle under investigation. The supernormality of the excitability grows from the demyelinated fibre without reorganized membrane to the demyelinated fibre with reorganized one. For short interstimulus intervals, the second action potential propagates along the fibres with a reduced velocity and a decreased amplitude. No supernormality of the potential parameters (amplitude, velocity) is observed during the cycle up to 30 ms. The membrane properties of the myelinated and demyelinated fibres with blocked potassium channels recover in the interval from 15 to 20 ms depending on whether the sodium channels' increase of the permeability is added on the background of the blocked potassium channel or not. In the recovery cycle, the axonal membrane reorganization leads to an improvement of the conduction along most severely demyelinated fibres.     

Different effects of blocked potassium channels on action potentials, accommodation, adaptation and anode break excitation in human motor and sensory myelinated nerve fibres: computer simulations

 Action potentials and electrotonic responses to 300-ms depolarizing and hyperpolarizing currents for human motor and sensory myelinated nerve fibres have been simulated on the basis of double cable models. The effects of blocked nodal or internodal potassium (fast or slow) channels on the fibre action potentials, early and late adaptations to 30-ms suprathreshold slowly increasing depolarizing stimuli have been examined. The effects of the same channels on accommodation after the termination of a prolonged (100 ms) hyperpolarizing current pulse have also been investigated. By removing the nodal fast potassium conductance the action potentials of the sensory fibres are considerably broader than those of the motor neurons. For both types of fibres, the blocked nodal slow potassium channels have a substantially smaller effect on the action potential repolarization. When the suprathreshold depolarizing current intensity is increased, the onset of the spike burst occurs sooner, which is common in the behaviour of the fibres. The most striking differences in the burst activity during early adaptation have been found between the fibres when the nodal fast potassium channels are blocked. The results obtained confirm the fact that the motor fibres adapt more quickly to sustained depolarizing current pulses than the sensory ones. The results also show that normal human motor and sensory fibres cannot be excited by a 100-ms hyperpolarizing current pulse, even at the threshold level. When removing the potassium channels in the nodal or internodal axolemma, the posthyperpolarization increase in excitability is small, which is common in the behaviour of the fibres. However, anode break excitation can be simulated in the fibres with simultaneous removal of the potassium channels under the myelin sheath, and this is more pronounced in the human sensory fibres than in motor fibres. This phenomenon can also be found when the internodal and some of the nodal (fast or slow) potassium channels are simultaneously blocked. Received: 8 November 1999 / Accepted in revised form: 29 February 2000     

Peripheral nerve at extreme low temperatures 2: Pharmacologic modulation of temperature effects

Changes in temperature have profound and clinically important effects on the peripheral nerve. In a previous paper, the effects of temperature on many properties of the peripheral nerve action potential (NAP) were explored including the NAP amplitude, conduction velocity and response to paired pulse stimulation. In this paper, the effects of pharmacologic manipulations on these parameters were explored in order to further understand the mechanisms of these effects.The reduction in conduction velocity with temperature was shown to be independent of the ionic composition of the perfusate and was unaffected by potassium or sodium channel blockade. This implies that the phenomenon of reduced conduction velocities at low temperature may be related to changes in the passive properties of the axon with temperature. Blockade of sodium channels and chronic membrane depolarization produced by high perfusate potassium concentrations or high dose 4-aminopyridine impair the resistance of the nerve to hypothermia and enhance the injury to the nerve produced by cycles of cooling and rewarming. This suggests the possibility that changes in the sodium inactivation channel may be responsible for the changes in the NAP amplitude with temperature and that prolonged sodium inactivation may lead more permanent changes in excitability.     

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization,veratridine, or A23187 in rat cerebral cortical synaptosomes

The effect of 4-aminopyridine on [³H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [³H]acetylcholine release in a concentration-dependent manner (5–10 mM); a high concentration (10 mM) also elevated [³H]choline efflux. However, the 35 mM K⁺ induced release of [³H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K⁺ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 M). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K⁺ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.     

Ion Channel Clustering at the Axon Initial Segment and Node of Ranvier Evolved Sequentially in Early Chordates

In many mammalian neurons, dense clusters of ion channels at the axonal initial segment and nodes of Ranvier underlie action potential generation and rapid conduction. Axonal clustering of mammalian voltage-gated sodium and KCNQ (Kv7) potassium channels is based on linkage to the actin–spectrin cytoskeleton, which is mediated by the adaptor protein ankyrin-G. We identified key steps in the evolution of this axonal channel clustering. The anchor motif for sodium channel clustering evolved early in the chordate lineage before the divergence of the wormlike cephalochordate, amphioxus. Axons of the lamprey, a very primitive vertebrate, exhibited some invertebrate features (lack of myelin, use of giant diameter to hasten conduction), but possessed narrow initial segments bearing sodium channel clusters like in more recently evolved vertebrates. The KCNQ potassium channel anchor motif evolved after the divergence of lampreys from other vertebrates, in a common ancestor of shark and humans. Thus, clustering of voltage-gated sodium channels was a pivotal early innovation of the chordates. Sodium channel clusters at the axon initial segment serving the generation of action potentials evolved long before the node of Ranvier. KCNQ channels acquired anchors allowing their integration into pre-existing sodium channel complexes at about the same time that ancient vertebrates acquired myelin, saltatory conduction, and hinged jaws. The early chordate refinements in action potential mechanisms we have elucidated appear essential to the complex neural signaling, active behavior, and evolutionary success of vertebrates.     

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Quinidine blocking of sodium and potassium channels in myelinated nerve fibers

Experiments by the voltage clamp method showed that external application of quinidine (5 × 10^–5 M) to the Ranvier node membrane of the frog nerve fiber inhibitis both sodium and potassium currents. Blocking of the sodium current is considerably intensified by repetitive depolarization of the membrane (1–10 Hz); the rate of development of the block increases with an increase in stimulation frequency. After the end of stimulation the sodium current gradually returns to its initial level (with a time constant of the order of 30 sec at 12°C). Unlike repetitive depolarization with short (5 msec) stimuli, a prolonged shift (1 sec) of potential toward depolarization has no significant effect on quinidine blocking of the sodium current. Analysis of the current-voltage characteristic curves showed that quinidine blocks outward sodium current more strongly than inward. Batrachotoxin protects sodium channels against the blocking action of quinidine in a concentration of 10^–5 M. Inhibition of the outward potassium currents by quinidine is distinctly time-dependent in character: Initially the potassium current rises to a maximum, then falls steadily to a new stationary level. The results agree with the view that quinidine, applied externally, penetrates through the membrane in the basic form and blocks open sodium and potassium channels from within in the charged (protonated) form. The similarity in principle between the action of quinidine and local anesthetics on the sodium suggests that these compounds bind with the same receptor, located in the inner mouth of the sodium channel.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 324–330, May–June, 1982.     

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     

Gating currents in the node of Ranvier: voltage and time dependence.

Like the axolemma of the giant nerve fibre of the squid, the nodal membrane of frog myelinated nerve fibres after blocking transmembrane ionic currents exhibits asymmetrical displacement currents during and after hyperpolarizing and depolarizing voltage clamp pulses of equal size. The steady-state distribution of charges as a function of membrane potential is consistent with Boltzmanns law (midpoint potential minus 33.7 mV; saturation value 17200 charges/mum-2). The time course of the asymmetry current and the voltage dependence of its time constant are consistent with the notion that due to a sudden change in membrane potential the charges undergo a first order transition between two configurations. Size and voltage dependence of the time constant are similar to those of the activation of the sodium conductance assuming m-2h kinetics. The results suggest that the presence of ten times more sodium channels (5000/mum-2) in the node of Ranvier than in the squid giant axon with similar sodium conductance per channel (2-3 pS).     

Ionic channel distribution in regenerating mouse motor endings

One of the nerves supplying the Triangularis sterni muscle of the adult mouse was crushed and focal extracellular recordings were made from the regenerating motor nerve terminals. Stimulation of the nerve elicited 4 types of signals depending upon the position of the electrode along the newly formed terminal branches. Specific ionic channel blockers were applied in the bath or iontophoretically to identify the nature of the different signal components. Sodium and potassium channels appeared to be located along most of the length of the newly formed terminals. This overlapping distribution differs considerably from their segregated localization previously described for mature endings. During maturation of regenerating endings, potassium channels disappear from the heminodal area and the density of sodium channels is greatly reduced along the remainder of the branches. Presynaptic evidence for polyneuronal reinnervation of single endplates is presented.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na+ currents and Na+-current fluctuations were measured in myelinated frog nerve fibres at 15 degrees C during 7.7 ms depolarizations to V = 40, 60 and 80 mV. (2) The conductance gamma of a single Na+ channel and the number No of channels per node were calculated from ensemble average values of the mean Na+ current and the variance of Na+-current fluctuations. (3) For a hyperpolarizing holding potential of VH = -28 mV the mean values of the channel conductance and number were gamma = 9.8 pS and No = 74000. (4) After changing the holding potential to the resting potential (VH = 0) the conductance gamma increased by a factor of 1.37 whereas the number No decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of VH = -28 mV increased gamma by a factor of 1.55 and reduced No by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na+ channels in the nodal membrane.     

Anomalous response of rabbit papillary muscles to depolarising current: the possible role of the transient outward current. A pharmacological analysis

Rabbit papillary muscles under current depolarization generate an anomalous double action potential (AP) instead of a usual repetitive activity characteristic of myocardial fibres of different mammalian species. The mechanism of the double AP consisting of a spike-like and a delayed slow component was analysed using pharmacological approach. No changes in the anomalous double AP were observed in the presence of Cs ions. This contrasted with the inhibitory action of 4-aminopyridine (4-Apy). High sensitivity of the phenomenon to 4-APy suggests a contribution of the transient outward current, previously postulated for rabbit working myocardial fibres, to account for the double AP.     

Mechanisms of action of voltage-gated sodium channel ligands

The voltage-gated sodium channels play a key role in the generation of action potential in excitable cells. Sodium channels are targeted by a number of modulating ligands. Despite numerous studies, the mechanisms of action of many ligands are still unknown. The main cause of the problem is the absence of the channel structure. Sodium channels belong to the superfamily of P-loop channels that also the data abowt includes potassium and calcium channels and the channels of ionotropic glutamate receptors. Crystallization of several potassium channels has opened a possibility to analyze the structure of other members of the superfamily using the homology modeling approach. The present study summarizes the results of several recent modelling studies of such sodium channel ligands as tetrodotoxin, batrachotoxin and local anesthetics. Comparison of available experimental data with X-ray structures of potassium channels has provided a new level of understanding of the mechanisms of action of sodium channel ligands and has allowed proposing several testable hypotheses.     

Ciguatoxins and brevetoxins: dissection of the neurobiological actions]

This review focuses on the neurobiological actions of ciguatoxins and brevetoxins which are phycotoxins produced respectively by the dinoflagellates Gambierdiscus toxicus and Ptychodiscus brevis. These actions are illustrated in particular by the effects of the toxins on myelinated nerve fibres and on skeletal neuromuscular junctions of vertebrates. Ciguatoxins and brevetoxins, through different vectors, are responsible for human intoxications characterized mainly by neurological disturbances. The molecular target of these families of lipid-soluble cyclic polyethers is the voltage-gated sodium channel, a fundamental transmembrane protein involved in cellular excitability. The different toxins share a common binding site (the receptor-site 5) located on the alpha sub-unit of this neuronal transmembrane protein. Electrophysiological studies of the mode of action of ciguatoxins and brevetoxins identify these toxins as specific sodium channel activators. Indeed, during the action of these phycotoxins, sodium channels remain permanently opened, at the resting membrane potential, which produces a continuous entry of sodium ions in most excitable cells. Such a sodium entry has various consequences on sodium-dependent physiological mechanisms, consisting in a membrane depolarization which, in turn, causes spontaneous and/or repetitive action potential discharges and thereby increases membrane excitability. These neuronal discharges may be transient or continuous according to the preparation and the toxin tested. The increase in membrane excitability during the action of ciguatoxins and brevetoxins is responsible for the different effects exerted by these toxins on various chemical synapses and secretory cells. Another consequence of the continuous entry of sodium ions into cells was revealed using confocal laser scanning microscopy and vital staining of plasma membranes with the fluorescent dye FM1-43. These techniques made feasible the dynamic study of morphological alterations produced by ciguatoxins and brevetoxins on various cellular preparations in situ. Thus, it has been possible to bring to the fore that these phycotoxins cause a marked increase in the volume of nodes of Ranvier of myelinated nerve fibres, motor nerve terminals innervating skeletal muscle and perisynaptic non-myelinating Schwann cell somata. This increase could be reversed by hyperosmotic external solutions and completely prevented by the blockade of voltage-gated sodium channels. The mechanisms involved in the increase in cellular volume, during the action of ciguatoxins and brevetoxins, are discussed.     

Action potential refractory period in axonal demyelination: a computer simulation

Axonal demyelination leads to an increase in the refractory period for propagation of the action potential. Computer simulations were used to investigate the mechanism by which changes in the passive properties of the internodal membrane increase the refractory period. The properties of the voltage dependent ion channels can be altered to restore conduction in demyeliated nerve fibers. The ability of these alterations to decrease the refractory period of demyelinated model nerve fibers was compared. The model nerve fiber contained six nodes. The action potential was stimulated at node one and propagated to node six. The internode between nodes three and four was demyelinated in a graded manner. The absolute refractory period for propagation of the action potential through the demyelinated internode increased as the number of myelin wraps was reduced to less than 25% of the normal value. The increase in refractory period was found to be due to a reduction in the rate or repolarization of the action potential at node three. The delay in repolarization reduced the rate of recovery of inactivated Na channels and slowed the closing of K channels. The rate of repolarization of node three was reduced by the conduction delay for the depolarization of node four caused by demyelination of the preceeding internode. In these simulations the increase in refractory period due to demyelination was eliminated by slowing the onset of Na channel inactivation. A small reduction of the K conductance also decreased the refractory period. However, larger reductions eliminated this effect.     

Sodium and potassium currents recorded during an action potential

A simple method was used to measure directly sodium and potassium currents underlying the action potential in single nerve fibres of Xenopus laevis. A short rectangular stimulus under current-clamp conditions elicited an action potential which was digitally stored and later used as command when voltageclamping the same fibre. The currents thus obtained nearly reproduced the original rectangular stimulus. Adding first 100 nM TTX and subsequently 100 nM TTX plus 10 mM TEA to the extracellular Ringer solution revealed the sodium and the potassium currents during an action potential. They were converted to permeabilities by use of the constant-field equation and are in good agreement with the curves which had been calculated from conventional voltage-clamp data. Thus experimentally determined currents and permeabilities are shown as they are changing during an action potential.