Sodium and potassium channels in regenerating and developing mammalian myelinated nerves

A study has been made of how the normal complementary distribution of sodium and potassium channels in mammalian myelinated nerve fibres (all the sodium channels being in the node with all the potassium channels in the internode) is altered in regenerating and in developing rabbit sciatic nerves. In regenerating nerve fibres, where a marked increase in the number of nodes per unit length occurs, there is a corresponding increase in the sodium channel content (determined from the maximum saturable binding of labelled saxitoxin), consistent with the idea that the number of sodium channels per node remains roughly constant. The use of 4-aminopyridine, which by blocking potassium channels prolongs the action potential, has shown that both in regenerating nerve fibres and in developing nerve fibres potassium currents contribute to the mammalian action potential. In both cases, with the passage of time, the sensitivity to 4-aminopyridine progressively decreases.     

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.     

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.     

Effects of some potassium channel blockers on the ionic currents in myelinated nerve

The effects of some potassium channel blockers on the ionic currents and on the so-called K(+)-depolarization in intact myelinated nerve fibres were studied. 4-AP, and in particular, Flaxedil, proved to be selective K(+)-current blockers. However, TEA, a crown ether (DCH18C6), a longchained triethylammonium compound (C10-TriEA), capsaicin, and the extract from the medicinal herb Ruta graveolens proved not to be selective K(+)-current blockers; they all block Na(+)-currents as well, although to a lesser extent. The sodium inactivation curve did not change under TEA and Flaxedil but was shifted on the potential axis in negative direction by DCH18C6, 4-AP, capsaicin and the Ruta extract whereas C10-TriEA caused a shift of both sodium inactivation and activation parameters in positive direction. Regarding to the kinetics of the persisting K(+)-current fraction, two different kinds of blockade were found: 1. Unchanged K(+)-kinetic which is typical for the effects of TEA, 4-AP, Flaxedil, and C10-TriEA. 2. Clearly changed K(+)-kinetic, characterized by K(+)-transients; which is typical for the effects of capsaicin and in particular, for those of DCH18C6 and of the Ruta extract. The possibly different modes of action of both groups of blockers are discussed in terms of current models for the action of potassium channel blockers.     

Local anaesthetic block protects against electrically-induced damage in peripheral nerve

This study is one of a series addressing the mechanisms involved in the production of neural damage caused by continuous, prolonged electrical stimulation of peripheral nerve. It has been previously shown that sustained, high frequency electrical stimulation of the cat's peroneal nerve may cause irreversible neural damage in the form of axonal degeneration of the large myelinated fibres. In this study we demonstrate that blocking the action potentials on most of the nerve fibres with local anaesthetics (10% procaine or 2% lidocaine) almost completely prevents the axonal degeneration. The abolition of axonal injury by local anaesthetic block strongly suggests that the electrically-induced damage is due to prolonged electrical excitation of axons. Furthermore, since less than complete suppression of the induced neural activity by local anaesthetic engenders essentially complete sparing of all axons, our results suggest that the damage to individual axons derives, at least in part, from stimulation-induced global changes in the nerve.     

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.     

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.     

Scanning electron microscopy of isolated peripheral nerve fibres

Summary The surface morphology of normal myelinated nerve fibres prepared in different ways for scanning electron microscopy has been studied and compared with the surface features of similar fibres undergoing retrograde changes. Nodes of Ranvier, paranodal specializations, artefactual fractures of the myelin, and the endoneurial collagen sheaths are described. A regular pattern of elevations, usually with a pitted or depressed surface seen on normal myelinated fibres after certain preparative procedures are thought to be artefacts produced during preparation and to be related to the neurokeratin network.Alterations in the surface structure of fibres central to long-standing nerve transections include irregular protuberances, serial surface corrugations and large swellings, all associated with demyelination. Fibres that have undergone retrograde degeneration consist of endoneurial tubes with focal swellings occupied by macrophages or myelin debris, together with fine unmyelinated and small myelinated regenerating axons. Strict centrifugal progression of myelination of regenerating axons was not observed.We thank Mr. R. A. Willis for his collaboration and for taking the SEM photographs of normal nerve fibres, and the Cambridge Scientific Instrument Co. Ltd. for permission to reproduce the SEM photographs of experimental nerve fibres. We also thank Dr. A. Boyde for access to his SEM and for helpful comments on interpretation of the scanning electron micrographs, Prof. J. Z. Young, Dr. P. K. Thomas, and Dr. R. H. M. King for discussion, and Messrs. P. Reynolds and D. Gunn for photography.A grant from the Muscular Dystrophy Group of Great Britain is gratefully acknowledged.     

Study of effect of embryonic anlage allografts of the rat spinal cord on growth of regenerating fibers of the recipient nerve

A comparative study of the effect of tissue and suspension allografts of an embryonic spinal cord on regeneration of nerve fibers of impaired (by application of a ligature) sciatic nerve in rats was conducted. It was demonstrated that unlike tissue grafts that reach a large volume 21 and 60 days after transplantation, suspension grafts do not inhibit the growth of axons of the recipient to the periphery. It was established that introduction of a suspension of dissociated cells of the spinal cord embryonic anlages (but not fragments of these anlages) into the impaired sciatic nerve in rats results in an increase in the amount of myelinated regenerating nerve fibers of the recipient 60 days after the operation.     

Neurogenic inflammation of gingivomucosal tissue in streptozotocin-diabetic rat

Previously it was assumed that nerve fibres are involved in the neurogenic inflammation induced by mechanical or chemical irriations. It has been also suggested that in diabetes mellitus the unmyelinated small diameter fibers are impaired as a result of diabetic neuropathy. Therefore, our aim was to study the alterations of the nerve processes in the gingivomucosal tissue in streptozotocin (STZ)-diabetic rats. Light- and electronmicroscopical examinations were made to analyze the changes in nerve fibres. After one week of steptozotocin treatment, the gingivomucosal tissue had inflammatory cell infiltration and some degenerated nerve fibres were also observed. Dense mitochondria, disorganization of cell organelles, and appearance of myelin-like dense bodies were found in the axons of degenerared nerve fibres. Semiquantitative analysis showed that 14 +/- 4% of the unmyelinated nerve fibres degenerated after one week of STZ treatment. However, degeneration of the myelinated nerve fibers was not observed. Two weeks after STZ treatment, most of the unmyelinated and myelinated nerve fibers showed degeneration (86 +/- 5%) and the placement of the ligature revealed a non-inflammatory connective tissue adjacent to a normal epithelium. The myelin sheath was disrupted and dark axoplasm with cytolysosomes became manifest. These findings demonstrated that both unmyelinated and myelinated nerve fibers are altered and inflammatory reaction exists in the gingivomucosal tissue only in the early stage of diabetes mellitus.     

The freezing threshold of the peripheral motor nerve: an electrophysiological and light-microscopical study on the sciatic nerve of the rabbit.

Fifty sciatic nerves of 39 rabbits are treated at different temperatures (+5, +1, 0, ?3, ?5, ?10, ?15 and ?20 °C), for different freezing times (10, 20, 30, 60 and 120 sec), and for different numbers of freeze-thaw cycles (1, 2 and 4). After electric supramaximal stimulation (3.8 V) action potentials of the sciatic nerve are measured before, immediately after, and 1, 3, 5, 10, 20, 30, 60, 90 min, 2, 5 and 10 days after freezing. Two or ten days after freezing, the nerves are examined in a light microscope. The cold threshold of the sciatic nerve was determined, i.e., the temperature at which after supramaximal stimulation it is still possible to measure an action potential within 1.5 hr after freezing. On application of one freeze-thaw cycle, the cold threshold is ?15 °C after a freezing time of 10 sec, ?10 °C after 20 sec and 30 sec, and ?5 °C after 60 and 120 sec. After application of two and four freeze-thaw cycles, the cold threshold is elevated, and after a super-cooling time of 10 sec it is ?10 °C, after 30 sec ?5 °C. The longer the freezing time and the more freeze-thaw cycles, the higher is the cold threshold. At ?20 °C (superthreshold temperature) an action potential can no longer be measured and all myelinated nerve fibres have decayed, except some small-caliber ones.Electrophysiologically, it is evident that some of the myelinated nerve fibres become functionally damaged for 1.5 hr, while other parts of the nerve fibres will degenerate and later regenerate. The amplitudes of the measured action potentials correlate with the decay of myelin sheaths and axons of large- and medium-caliber nerve fibres. Action potentials between 0 and 40% show a gradual paresis, above 40% a physiological motor function. The pathophysiological mechanism of this reversible functional loss after super-cooling and freezing may be a consequence of a disturbed membrane permeability.It is of clinical importance that, if the cold threshold of a peripheral motor nerve is known, the nerve can be frozen concomitantly for a short time at application of low temperatures without suffering any functional loss. This is achieved by controlling during freezing the motor function of the corresponding nerve situated on the periphery of cryolesion, and, if there is a loss of motor function, the freezing process has to be interrupted immediately.     

Effect of vitamin E-deficiency on regeneration of the sciatic nerve

The regeneration of the sciatic nerve fibres was studied in both normal and vitamin E-deficient rats at 30 and 60 days after crush. The vitamin E is involved in one of the most important mechanisms of protection against peroxidation of plasma membrane lipids; the plasma membrane plays certainly a role in nerve regeneration. Both the diameter and the total number of myelinated nerve fibres was calculated at different times. The number of myelinated fibres in the undenervated deficient animals was lower than that found in the undenervated normals animals. Following the nerve crush, in normal animals after two months the number of myelinated fibres exceeded the number found in undenervated normal animals, whereas in the deficient rat nerves it was significantly lower than in the corresponding controls and moreover it did not even reach the number found in the nerves of undenervated deficient rats. Finally, the caliber distribution of myelinated fibres in undenervated and denervated deficient rats shows a relative percent increase in the number of greatest axons and a decrease in smaller axons. This result confirm the vitamin E to be an important factor of the normal process of nerve regeneration.     

Further studies on the functional properties of spinal axons in vivo

Mammalian spinal tracts in situ demonstrate a phase of marked hyperexcitability during hypoxia or on the application of an excess of potassium or citrate ion. This is in keeping with the fact that they also show post-spike supernormality as well as hyperexcitability under cathodal polarization (17). Behavior of this kind indicates that central axons carry a well developed L fraction of membrane properties. The rhythmic state in central axons in situ, unlike peripheral nerve or spinal root, is not induced by the action of excess potassium ion. This appears to be related to the absence of a positive after-potential in dorsal columns (17). However, sodium citrate can elicit autonomous firing in central axons. When synchronized by an applied stimulus the resulting periodic oscillations have a fundamental frequency (340 to 400 C.P.S.) which is significantly greater than that of peripheral nerve.     

Interaction of internal anions with potassium channels of the squid giant axon

The interaction of internal anions with the delayed rectifier potassium channel was studied in perfused squid axons. Changing the internal potassium salt from K+ glutamate- to KF produced a reversible decline of outward K currents and a marked slowing of the activation of K channels at all voltages. Fluoride ions exert a differential effect upon K channel gating kinetics whereby activation of IK during depolarizing steps is slowed dramatically, but the rate of closing after the step is not much altered. These effects develop with a slow time course (30-60 min) and are specific for K channels over Na channels. Both the amplitude and activation rate of IK were restored within seconds upon return to internal glutamate solutions. The fluoride effect is independent of the external K+ concentration and test membrane potential, and does not recover with repetitive application of depolarizing voltage steps. Of 11 different anions tested, all inorganic species induced similar decreases and slowing of IK, while K currents were maintained during extended perfusion with several organic anions. Anions do not alter the reversal potential or shape of the instantaneous current-voltage relation of open K channels. The effect of prolonged exposure to internal fluoride could be partially reversed by the addition of cationic K channel blocking agents such as TEA+, 4-AP+, and Cs+. The competitive antagonism between inorganic anions and internal cationic K channel blockers suggests that they may interact at a related site(s). These results indicate that inorganic anions modify part of the K channel gating mechanism (activation) at a locus near the inner channel surface.     

Blockade of sodium and potassium channels in the node of Ranvier by ajmaline and N-propyl ajmaline

The inhibition of sodium and potassium currents in frog myelinated fibres by ajmaline (AM) and its quaternary derivative, N-propyl ajmaline (NPA), depends on voltage-clamp pulses and the state of channel gating mechanisms. The permanently charged NPA and protonated AM interact only (or mainly) with open channels, while unprotonated AM affects preferently inactivated Na channels. Inhibition of Na currents by NPA and AM does not depend on the current direction and Na ion concentration in external or internal media. In contrast only the outward potassium currents can be blocked by NPA and AM; the inward potassium currents in high K+ ions external media are resistant to the blocking action of these drugs. The voltage dependence of ionic current inhibition by charged drugs suggests the location of their binding sites in the inner mouths of Na and K channels. Judging by the kinetics of current restoration after cessation of pulsing, the drug-binding site complex is much more stable in Na than in potassium channels. Batrachotoxin and aconitine, unlike veratridine and sea anemone toxin, decrease greatly the affinity of Na channel binding sites to NPA and AM. The effects of NPA and AM are compared with those of local anesthetics and other amine blocking drugs.     

The action potential of spinal axons in vitro

Despite the trauma of dissection and special metabolic requirements, the physiological properties of funiculi of the mammalian spinal cord can be studied in vitro. They are adequately oxygenated by diffusion at 0.88 atm. pO(2) and remain in a functionally normal state for over 12 hours. The internal consistency of several kinds of data presented in this and the foregoing papers (5, 38) serves to characterize certain properties of central myelinated axons whether excised or in situ. (1) Spinal tracts support a large spike potential in vitro whose form, duration, and velocity are comparable to those of alpha fibers in vitro and spinal tracts in vivo. (2) Properties consistent with a large L fraction are found in central axons whether excised or in situ. (3) Following conduction there has been identified post-spike supernormality with exponential time course (7.5 msecs. half-time) which is the result of activity intrinsic to parent fibers of dorsal columns. The supernormality is similar in form and magnitude both in excised and intact funiculi. (4) In excised funiculi the action potential of parent axons includes a large negative after-potential whose form and duration correspond satisfactorily with this supernormality. This potential appears not to result from activity arising in broken collaterals. (5) Central axons, excised or intact, fire spontaneously in the presence of citrate ion, and when synchronized by stimulation develop periodic oscillations at about 400 C.P.S. but show no such behavior in the presence of excess potassium ion. Certain characteristics peculiar to central axons indicate that they occupy an extreme position in the spectrum of properties encountered in conducting tissues. Dorsal column myelinated axons differ from their peripheral counterparts, even though they are parts of the same cell, in the following ways. The maintenance of the column spike potential is more critically dependent on CO(2) and the entire tissue mass has a higher oxygen consumption. The negative after-potential is much larger and the positive after-potential, non-existent following a single volley, is more difficult to develop by repetitive stimulation. Unlike peripheral nerve, central axons are not incited to spontaneous activity by manipulation of certain constituents normally present in their environment. However, when induced by the application of citrate the resulting rhythmic behavior has twice the frequency of that in peripheral nerve. In general, the recovery process in central axons is more invariant than that in peripheral axons when they are subjected to similar changes in their artificial environments.     

Epineurial Window Is More Efficient in Attracting Axons than Simple Coaptation in a Sutureless (Cyanoacrylate-Bound) Model of End-to-Side Nerve Repair in the Rat Upper Limb: Functional and Morphometric Evidences and Review of the Literature

End-to-side nerve coaptation brings regenerating axons from the donor to the recipient nerve. Several techniques have been used to perform coaptation: microsurgical sutures with and without opening a window into the epi(peri)neurial connective tissue; among these, window techniques have been proven more effective in inducing axonal regeneration. The authors developed a sutureless model of end-to-side coaptation in the rat upper limb. In 19 adult Wistar rats, the median and the ulnar nerves of the left arm were approached from the axillary region, the median nerve transected and the proximal stump sutured to the pectoral muscle to prevent regeneration. Animals were then randomly divided in two experimental groups (7 animals each, 5 animals acting as control): Group 1: the distal stump of the transected median nerve was fixed to the ulnar nerve by applying cyanoacrylate solution; Group 2: a small epineurial window was opened into the epineurium of the ulnar nerve, caring to avoid damage to the nerve fibres; the distal stump of the transected median nerve was then fixed to the ulnar nerve by applying cyanoacrylate solution. The grasping test for functional evaluation was repeated every 10–11 weeks starting from week-15, up to the sacrifice (week 36). At week 36, the animals were sacrificed and the regenerated nerves harvested and processed for morphological investigations (high-resolution light microscopy as well as stereological and morphometrical analysis). This study shows that a) cyanoacrylate in end-to-side coaptation produces scarless axon regeneration without toxic effects; b) axonal regeneration and myelination occur even without opening an epineurial window, but c) the window is related to a larger number of regenerating fibres, especially myelinated and mature, and better functional outcomes.