Conduction failure in myelinated and non-myelinated axons at low temperatures

1. The effects of low temperature on conduction in single myelinated and non-myelinated axons of the feline saphenous nerve were examined and compared. Nerves were cooled by a conventional thermode, but thermal gradients were minimized by an insulating layer of agar-saline gel over the nerve and the face of the thermode.2. The mean blocking temperature of thirty-one non-myelinated axons, 2.7 degrees C, was significantly lower than that of 111 myelinated axons, 7.2 degrees C. No evidence for a differential block of myelinated axons according to their normal conduction velocity could be demonstrated.3. Reductions in the proportional conduction velocities of both myelinated and non-myelinated axons were nearly identical between 17 and 37 degrees C. However, below 17 degrees C the rate at which the proportional conduction velocity of the non-myelinated axons fell during cooling was significantly less than for the myelinated axons and was sufficient to account for their lower blocking temperatures. As a result, critical minimum conduction velocities were reached at higher temperatures in myelinated axons than in non-myelinated axons.4. The conduction velocity of successive impulses in a train slowed progressively to a constant value which depended on the frequency of stimulation. Consequently, the early impulses were separated by intervals that exceeded those between stimuli and were not affected by temperatures that blocked later impulses. The pattern of block was consistent with an increasing refractoriness of the axons as the temperature fell.5. The maximal frequency of discharge that myelinated axons could carry at temperatures between normal and 12 degrees C was related directly to fibre size. Non-myelinated axons could conduct low frequency trains of impulses at temperatures that blocked such activity in myelinated axons. In all axons, high frequency trains of impulses could be completely blocked at temperatures which permitted lower frequency trains to pass uninterrupted.6. Hysteresis in the blocking temperatures of axons was related to the hysteresis in their conduction velocities.     

Distribution and morphological characteristics of axons in the periodontal ligament of cat canine teeth and the changes observed after reinnervation

The distribution and morphological characteristics of myelinated and non-myelinated axons innervating the lower canine periodontal ligament (PDL) in adult cats have been analysed. After perfusion fixation and decalcification, the teeth were slit transversely, divided into segments, and embedded in plastic. Ultrathin sections of each segment were examined in the electron microscope and used to reconstruct the whole PDL at 1, 4, 7, and 9 mm from the tooth apex. One millimeter from the tooth apex there were a mean of 920 myelinated axons and 1,415 non-myelinated axons. The numbers of axons declined toward the tooth crown. Bundles of myelinated and small non-myelinated axons lay adjacent to the blood vessels midway between the bone and cementum. Isolated myelinated axons appeared to have split away from these main nerve bundles and entered the avascular zone of the ligament, where they lost their myelin sheaths to become large non-myelinated axons rich in mitochondria. These non-myelinated axons sometimes appeared to be linked to collagen fibres and were thought to be the mechanoreceptor terminals. Twelve weeks after sectioning and inferior alveolar nerve, the total number of axons innervating the periodontal ligament was 50% of that found in the contralateral controls. The large non-myelinated axons had smaller mean diameters and contained fewer mitochondria, a change which may be consistent with a reduction in mechanoreceptor excitability.     

Observation on the localization of mechanoreceptors in the kidney and afferent nerve fibres in the renal nerves in the rabbit.

1. The distribution and localization of mechanoreceptors in the kidney were studied by recording afferent impulses from the renal nerve bundle or from single nerve fibres in the isoloted kidney preparation in the rabbit. 2. It was observed that mechanoreceptors are distributed in the cranial, central and caudal portions as well as the pelvic portion of the kidney. Diameter range of single nerve fibres from which afferent impulses were recorded was from 2 to 8 mum. 3. Histological studies show that the renal nerve possesses abundant non-myelinated nerve fibres with a relatively small number of myelinated nerve fibres. The myelinated axons had diameters ranging from 0-5 to 13-4 mum and the peak of the unimodal distribution curve was 1-5--2-4 mum.     

Occurrence of long non-myelinated axonal segments intercalated in myelinated, presumably sensory axons: electron microscopic observations in the dog atrial endocardium

Summary Electron microscopy of serial sections revealed the occurrence of long non-myelinated segments in myelinated, presumably sensory axons running in the left atrial endocardium of normal adult dogs. Four such non-myelinated segments were analysed in three myelinated axons. They varied from 20 to 150 m in length, and differed from nodes of Ranvier in being invested by Schwarm cells in the manner of unmyelinated nerve fibres. Short non-myelinated portions (20–25 m long) were associated with a single Schwann cell, whereas the longest such segment (150 gmm) had five. The non-myelinated axonal segments were non-varicose and similar in diameter (1.2–3.0 m) to adjacent myelinated segments, which had myelin sheaths 6–25 lamellae thick. The cytoplasm of the non-myelinated axonal segments contained numerous neurofilaments and microtubules, some mitochondria and smooth endoplasmic reticulum. The short non-myelinated segments were enclosed by perineurium, whereas the long non-myelinated segment was devoid of perineurium at its mid-portion; instead fibroblast-like cells made a loose boundary around the axon at this level. The significance of these non-myelinated segments was discussed with special emphasis on the question of whether they result from focal degeneration of the myelin sheath (demyelination) or are generally present in the preterminal regions of some axons.     

The role of non-myelinated vagal afferent fibres from the lungs in the genesis of tachypnoea in the rabbit

1. The use of a direct current (d.c.) to produce a differential block of conduction in the cervical vagus nerves of rabbits is described; the myelinated fibres are blocked, while the non-myelinated ;C' fibres conduct normally. The method produces reproducible and reversible results.2. The block is equally effective for low and high frequencies of discharge (1-100 Hz). During recovery or development of the block, lower frequencies of discharge can pass but higher frequencies cannot.3. Block of conduction in myelinated fibres is associated with slower, deeper breathing, confirming previous work with cooling.4. A further slowing and deepening of breathing may occur when a differentially blocked (;non-myelinated') nerve is sectioned, and this is mainly apparent when there are pathological conditions in the lungs.5. The respiratory response to the right atrial injection of phenyl diguanide is mediated by non-myelinated thoracic vagal afferent fibres.6. The tachypnoeic response to lung deflation is not mediated by non-myelinated fibres.7. Head's Paradoxical reflex has been demonstrated during partial recovery of conduction in myelinated fibres when only lower frequencies of afferent discharge can pass the area of block.8. A standard technique for providing a reproducible vagally mediated, tachypnoeic response to pulmonary micro-embolism is described using inert carbon-coated microspheres of 50 mum diameter. This tachypnoeic response was unchanged during a differential block indicating that the response was mediated by non-myelinated ;C' fibres.9. Pathological changes such as haemorrhage, oedema, infarction and collapse were absent after micro-embolism, and there were no systematic changes in lung resistance and compliance. The walls of arterioles and adjacent alveoli are distorted by the emboli and these areas are the probable sites of afferent stimulation.     

Selective vulnerability of unmyelinated fiber Schwann cells in nerves exposed to local anesthetics

When peripheral nerves of experimental rats are exposed to local anesthetics, distinctive and reproducible pathologic changes occur involving the perineurial sheath and endoneurial contents. Application of intermediate strength concentrations of the local anesthetics, 2-chloroprocaine, lidocaine, etidocaine, and intermediate or high concentrations of procaine to the surface of rat sciatic nerves resulted in the following changes. By 48 hours, the perineurial sheath exposed to the drug was disrupted and became permeable to granulocytes which infiltrated the subjacent endoneurium in conjunction with edema formation in the endoneurial interstitium. Application of 10% procaine to exposed nerve resulted in extensive demyelination. The most striking pathologic change occurring with either intermediate or high doses was accumulation of lipid droplets in Schwann cells, a phenomenon that occurred often in myelin-producing Schwann cells but much less frequently in unmyelinated fiber Schwann Cells. Lipid accumulation appears to be one of several reactive changes that affect Schwann cells of myelinated fibers and is dose-dependent. On the other hand, while reactive changes were infrequently seen in unmyelinated fiber Schwann cells, these cells appeared more susceptible to injury as shown by electron microscopy. Injury to Schwann cells by local anesthetics is temporary because these cells can replicate quickly. Autoradiographic studies of thymidine incorporation 1 week after procaine administration to the sciatic nerve showed intense proliferation of Schwann cells, but no such activity in controls. These findings support the view that their neurotoxic properties may account in some part for the function of local anesthetics, that Schwann cells of small unmyelinated fibers are more vulnerable to these agents than those of myelinated fibers, and that destruction of their supporting cells is followed by vigorous mitotic activity in the endoneurium.     

Morphology of the long and short uveal nerves in the human eye

The aim of this study was to examine the architecture of the uveal nerves in the sclera and suprachoroid of human eyes. Eyes from 17 adult human donors were investigated. The uveal nerves in different regions (retrobulbar, intrascleral, suprachoroidal, pars plana) were prepared and studied by light and electron microscopy. In addition, immunohistochemistry was performed for various neuronal markers. The long uveal nerves showed a characteristic suprachoroidal location with no branches supplying the choroid. It was found that typically they are composed of myelinated (75%) and non-myelinated (25%) nerve fibres. They mainly contain aminergic and sensory nerve fibres. A separate set of cholinergic non-myelinated nerve fibre bundles runs parallel with these long uveal nerves. The short uveal nerves supply the suprachoroidal nerve plexus with approximately 13% of their nerve fibres. The nerves and the branches supplying the choroid appear as mixed nerves containing sympathetic, parasympathetic and sensory axons. This study therefore provides new information about the quantity, type and distribution of myelinated and non-myelinated nerve fibres in the posterior uvea of the human eye.     

Selective Activation of peripheral nerve fibre groups of different diameter by triangular shaped stimulus pulses.

1. The differential block of cutaneous nerve fibres has been achieved with a simple method of electrical stimulation, employing a single pair of active electrodes. 2. The method allows the selective activation of 95% of small myelinated (delta) axons, without activation of the larger (beta) ones; and activation of unmyelinated (C) fibres, without A fibre activation. Asynchronous firing of myelinated axons was absent in the majority of the experiments. 3. The method employs triangularly shaped electrical pulses, with a steep rise front and a slow exponential decay. The outward flow of current at the cathode fires conducted impulses in both larger and smaller axons, and the inward flow inactivates differentially the conduction in the smaller ones. 4. The differential effect of anodal currents rests upon the greater internal conductance and greater conduction velocity of larger fibres. 5. The method has the advantage over the conventional polarization block of simpler surgical preparation, longer nerve survival and minimal latency distortion. However, it cannot be applied in experiments requiring physiological stimulation of peripheral receptors.     

Opioidergic inhibition of reflexes evoked by selective stimulation of sural nerve C fibres in the rabbit.

Reflexes were evoked in the gastrocnemius medialis (GM) muscle nerve by selective electrical stimulation of the non-myelinated C fibres of the ipsilateral sural nerve of decerebrated, spinalized rabbits. The opioid antagonist (-)-quadazocine (555 micrograms/kg i.v.) enhanced responses to sural C fibre stimulation to an average of 236% of pre-drug levels. In addition, C fibre-evoked reflexes were depressed for 7-9 min after repetitive activation of the high threshold axons of the common peroneal nerve, and this effect was reversed after quadazocine. Thus, GM responses to stimulation of non-myelinated sural afferent fibres are suppressed by endogenous opioid peptides, but the degree of inhibition does not appear to be as profound as that previously reported for reflexes evoked by myelinated fibres.     

Effects of nerve compression or ischaemia on conduction properties of myelinated and non-myelinated nerve fibres. An experimental study in the rabbit common peroneal nerve

Compound action potentials of both myelinated (A) and non-myelinated (C) fibres in the common peroneal nerve of rabbits were studied during and after acute, graded compression of the nerve at 200 or 400 mmHg applied for 2 h or during ischaemia created by nitrogen inhalation or aortic occlusion. Compression of the nerve at 200 mmHg blocked the AI component (large myelinated fibres) after about 23 min, while compression at 400 mmHg shortened this time to 11 min. The A2 component (thinner myelinated fibres) had a lower conduction velocity and a higher resistance to compression. There was just a slight decrease in conduction velocity of the non-myelinated fibres when the nerves were compressed at 200 mmHg for 2 h. However, compression at 400 mmHg for 2 h induced a marked deterioration of amplitude and conduction velocity of the C-fibres. There was an incomplete restitution of function of A- and C-fibres during 2 h of recovery. The thinner myelinated fibres were more susceptible to deprivation of oxygen than the thicker ones, while non-myelinated fibres differed in response according to method of ischaemia induction. It is concluded that non-myelinated fibres are very resistant to compression and a very high pressure (greater than 400 mmHg) is needed to affect these fibres.     

Morphometric analysis of the vagus nerve in non-diabetic and ketonuric diabetic Chinese hamsters

Morphometric analysis of axons from the ventral division of the vagus nerve of ketonuric diabetic Chinese hamsters and age-sex matched non-diabetic controls was performed to determine the frequency distribution and numerical and volume density. Myelinated fibres of diabetics displayed a significant reduction in diameter (P less than 0.001) compared with controls, which was correlated inversely with progressive ketonuria (P less than 0.05). The reduced calibre of myelinated fibres was the result of thin myelin sheaths rather than a reduction in axon diameter. A marked decrease in numerical density (P less than 0.05) and volume density (P less than 0.005) was found in the myelinated fibres of diabetics compared with controls. Non-myelinated axons showed a significant shift to smaller diameter (P less than 0.001) in diabetics, which was correlated inversely with duration of ketonuria (P less than 0.05). Numerical density of non-myelinated axons was increased (P less than 0.01) in diabetic hamsters whereas volume density was comparable in diabetic and control animals. These data provide morphological evidence of impairment in the parasympathetic nervous system which may be a major factor underlying previously reported gastrointestinal and pancreatic islet dysfunction in the diabetic Chinese hamster.     

Axons and glial interfaces: ultrastructural studies

At most vertebrate nerve transitional zones (TZs) there is a glial barrier which is pierced by axons passing between the CNS and PNS. Myelinated axons traverse this in individual tunnels. The same is true of larger non-myelinated axons. This holds widely among the vertebrates, for example, the large motor axons of the sea-lamprey Petromyzon (which also possess TZ specializations not found in mammals). Smaller non-myelinated axons traverse the TZ glial tunnels as fascicles and so the barriers are correspondingly less comprehensive for them. Accordingly, in nerves composed of non-myelinated axons, such as the vomeronasal or the olfactory, a TZ barrier stretching across the nerve is effectivelyabsent. The chordateAmphioxus differsfrom the vertebrates in lacking a TZ barrier throughout. Invertebrates also lack glial barriers at the TZs between ganglia and interconnecting nerve trunks. The glial barrier at the dorsal spinal root TZ (DRTZ) has considerable value for analysing protocols aimed at achieving CNS regeneration, because it provides a useful model of the gliotic reaction at sites of CNS injury. Also, it is especially amenable to morphometric analysis, and so enables objective quantification of different protocols. Being adjacent to the subarachnoid space, it is accessible for experimental intervention. The DRTZ was used to investigate the value of neurotrophin 3 (NT3) in promoting axon regeneration across the TZ barrier and into the CNS following dorsal root crush. It promoted extensive regeneration and vigorous non-myelinated axonal ensheathment. On average, around 40% of regenerating axons grew across the interface, compared with virtually none in its absence. These may have traversed the interface through loci occupied by axons prior to degeneration. Many regenerating axons became myelinated, both centrally and peripherally.     

Saxitoxin and procaine act independently on separate sites of the sodium channel

1. Voltage clamp experiments were done on single myelinated nerve fibres of the frog, Rana esculenta. 2. The time course of procaine action (1.0 mM at pH 7.2) was obtained from changes in INa on changing solutions during repetitive (1 HZ) depolarizing pulses of constant amplitude following hyperpolarizing prepulses. The mean half times of onset and offset of procaine block were 3.7 and 28 s, respectively. In the presence of 1.4 nM saxitoxin (STX) the corresponding times were virtually the same, 3.1 and 27 s. 3. Similarly, the time course of partial relief from procaine block that is obtained by increasing the frequency of the prepulse-test pulse pairs from 1-10 HZ was unaffected in the presence of STX. 4. Comparison of the equilibrium effects of procaine concentrations ranging from 0.03-1.0 mM suggest a one-to-one drug-receptor reaction. The fraction of Na channels blocked at equilibrium with 1.0 mM procaine, 1.4 nM STX, and 1.0 mM procaine + 1.4 nM STX was 0.81, 0.49, and 0.90, respectively. This result and the kinetic behaviour fully agree with the idea of two separate and independent receptors for procaine and STX.     

The binding of tetrodotoxin to nerve membranes

1. The% reduction in size of the externally recorded action potential produced by concentrations of tetrodotoxin (TTX) in the range 6-300 nM was determined for the small non-myelinated fibres of the rabbit cervical vagus nerve and of the walking leg nerves of crab and lobster. The concentration of TTX for 50% reduction was around 80 nM for rabbit vagus and 14 nM for crab nerve.2. Bio-assay procedures were devised to measure the amount of TTX taken up by a nerve when it was exposed to a very small volume of a solution whose TTX content was just great enough to produce 100% block of conduction. The extracellular space of each nerve was determined with [(14)C]sugar so that an allowance could be made for extracellular dilution.3. The TTX binding by rabbit, crab and lobster nerve was respectively 0.064, 0.053 and 0.036 p-mole/mg wet weight of nerve.4. The binding of saxitoxin was measured in rabbit vagus nerve, and found to be much the same as that of TTX.5. Control experiments on rabbit sciatic nerve, where the area of excitable membrane was much smaller, showed that there was relatively little unspecific binding of TTX.6. In view of the evidence presented here and elsewhere that the blocking of sodium conductance by TTX involves the attachment of only one TTX molecule at each sodium site, and that unspecific binding of TTX does not cause serious errors, these results suggest that in 1 mum(2) of nerve membrane the number of sodium sites is 75 for rabbit, 49 for crab, and 36 for lobster nerve.     

Tetrodotoxin block of A-fibre conduction and its effect on reflex responses evoked by electrical stimulation of the sural nerve in the decerebrated rabbit

In the present study, we have investigated the viability of using tetrodotoxin (TTX) to induce selective blockade of myelinated fibre conduction in rabbit sural nerve, and explored some aspects of reflexes evoked by non-myelinated sural nerve afferents before and after application of TTX. In rabbits decerebrated under halothane-nitrous oxide anaesthesia, application of 30 nM TTX to the desheathed sural nerve completely blocked Abeta and Adelta waves of the compound action potential evoked by electrical stimulation of the nerve at 95 times threshold. The amplitude of C-fibre volleys evoked by these stimuli was reduced to a mean of 60 % of pre-treatment values. Reflexes evoked in medial gastrocnemius motoneurones by sural nerve stimulation showed corresponding changes after TTX treatment, with activation latency increasing from 5-7 ms in the control state to > 100 ms after TTX application. Temporal summation (wind up) in long latency reflexes (> 100 ms) was significantly enhanced after application of TTX. These data show that low concentrations of TTX can selectively block conduction in rabbit sural nerve A-fibres, providing a method for studying the central actions of non-myelinated C-fibres in isolation.     

The early stages of wallerian degeneration in the severed optic nerve of the newt (Triturus viridescens)

The initiation of Wallerian degeneration in the severed optic nerve of the newt (Triturus viridescens) was very rapid and intense. Significant degeneration of nonmyelinated axons was observed as early as six hours after lesion (h.a.l.) and was almost complete by 48 h.a.l. Initial degeneration of non-myelinated axons began in “extracellular digestion chambers” formed between burgeoning ependymoglial processes. The remaining fragments and debris were later phagocytized by surrounding ependymoglial processes. Many axons of myelinated fibers have degenerated as early as 6 h.a.l. However, the overall population of myelinated axons degenerates at a much slower rate than nonmyelinated ones, for many of them appear intact as late as 48 h.a.l. Some myelin sheaths show significant signs of degeneration by 6 h.a.l. Indeed, by this time a number of myelinated fibers have completely degenerated leaving only large vacuolated spaces in the nerve parenchyma. Swelling and vacuolization of the sheath are among the earliest signs of myelin degeneration. The ependymoglial cell response to optic nerve lesion is manyfold and dramatic. By 6 h.a.l. there are signs of burgeoning ependymoglial processes which begin to resemble scar formation (gliosis) by 48 h.a.l. The morphological evidence is consistent with the concept of an important phagocytic role of ependymoglial cells during the early stages of optic nerve degeneration.     

Effects of nerve compression or ischaemia on conduction properties of myelinated and nonmyelinated nerve fibres. An experimental study in the rabbit common peroneal nerve

Compound action potentials of both myelinated (A) and non-myelinated (C) fibres in the common peroneal nerve of rabbits were studied during and after acute, graded compression of the nerve at 200 or 400 mmHg applied for 2 h or during ischaemia created by nitrogen inhalation or aortic occlusion. Compression of the nerve at 200 mmHg blocked the Ai component (large myelinated fibres) after about 23 min, while compression at 400 mmHg shortened this time to 11 min. The A2 component (thinner myelinated fibres) had a lower conduction velocity and a higher resistance to compression. There was just a slight decrease in conduction velocity of the nonmyelinated fibres when the nerves were compressed at 200 mmHg for 2 h. However, compression at 400 mmHg for 2 h induced a marked deterioration of amplitude and conduction velocity of the C-fibres. There was an incomplete restitution of function of A- and C-fibres during 2 h of recovery. The thinner myelinated fibres were more susceptible to deprivation of oxygen than the thicker ones, while non-myelinated fibres differed in response according to method of ischaemia induction. It is concluded that non-myelinated fibres are very resistant to compression and a very high pressure (> 400 mmHg) is needed to affect these fibres.     

Analysis of potassium channel functions in mammalian axons by gene knockouts

Mammalian axons express a rich repertoire of various K channel subtypes whose distribution is profoundly affected by myelination. In the past two decades, functional analysis of axonal K channels has been approached primarily through pharmacology. Recently, gene knockout techniques have been used to specifically delete a particular K channel subtype from axons. This is significant since the bulk of K channels in a myelinated nerve are covered by the myelin, making functional analysis of specific K channel subtypes by traditional means difficult. This review summarizes the first mutational analysis of this sort performed on an axonal fast K channel termed Kv1.1. This K channel is concealed by the myelin loops in the paranodes of all major myelinated fiber tracts, and exhibits highly heterogeneous distribution even in certain non-myelinated CNS axons. Physiological analysis of Kv1.1 null mutants suggest novel functions for this axonal K channel subtype, including modulation of conduction failures at branch points and stabilization of transition zones in myelinated nerves.     

Histomorphometric changes in repaired mouse sciatic nerves are unaffected by the application of a scar-reducing agent

Microsurgical repair of transected peripheral nerves is compromised by the formation of scar tissue and the development of a neuroma, thereby limiting the success of regeneration. The aim of this study was to quantify histomorphometrically the structural changes in neural tissue that result from repair, and determine the effect of mannose-6-phosphate (M6P), a scar-reducing agent previously shown to enhance regeneration. In anaesthetised C57-black-6 mice, the left sciatic nerve was sectioned and repaired using four epineurial sutures. Either 100 μL of 600 mm M6P (five animals) or 100 μL of phosphate-buffered saline (placebo controls, five animals) was injected into and around the nerve repair site. A further group acted as sham-operated controls. After recovery for 6 weeks, the nerve was harvested for analysis using light and electron microscopy. Analysis revealed that when compared with sham controls, myelinated axons had smaller diameters both proximal and distal to the repair. Myelinated axon counts, axonal density and size all decreased across the repair site. There were normal numbers and densities of non-myelinated axons both proximal and distal to the repair. However, there were more Remak bundles distal to the repair site, and fewer non-myelinated axons per Remak bundle. Application of M6P did not affect any of these parameters.