Myelin Layers Research Articles

The swelling property of central nervous system nerves using X-ray diffraction

Low-angle X-ray diffraction patterns have been recorded from cattle and rabbit optic nerves swollen in glycerol solutions. The new X-ray data have a resolution of about 15 to 16 Å. Analysis of the low-angle X-ray data indicates that the myelin layers of optic nerves swell in units of four membranes, that is, two membrane pairs adhere together during the process of swelling. Fourier syntheses of glycerol-treated cattle optic nerves are described. Differences in structure between the normal and swollen membrane pairs are apparent.

Zonulae occludentes of the myelin lamellae in the nerve fibre layer of the retina and in the optic nerve of the rabbit: a demonstration by the freeze-fracture method.

Ridges and grooves composing extensive zonulae occludentes are revealed by the freeze-fracture method on split myelin lamellae in the nerve fibre layer of the retina and in the optic nerve of the rabbit. The junctions are located immediately internal to the outer loop of the myelin sheath and in corresponding areas of deeper myelin layers. They follow a straight or gently undulating course along the axis of the fibres. Only at the paranodal region of nodes of Ranvier do they deviate and assume a transverse course, The strands of these zonulae occludentes probably represent the radial thickenings of the intraperiod line described in thin sections.

NEUROLOGY: EVIDENCE FOR THE PARTICIPATION OF A GLICOPROTEIN IN THE EARLY PROCESSES OF MYELINATION

The presence of a glycoprotein in myelin has been recently reported (Quarles et al.Biochem.biophys.Res.Commun.47, 491, 1972). Since glycoproteins on the plasma membrane of cells are believed to be involved in specific cell-to-cell interactions, the myelin associated glycoprotein could be involved in contact relationships between axons and oligodendrocytes or between the different layers of myelin. Myelin-associated glycoproteins were investigated during myelination in normal rats and in normal and myelin-deficient mutant mice. Glycoproteins were labelled in vivo by injection of [3H] or [14C] fucose, and purified myelin fractions were prepared. Proteins were extracted with SDS, separated by polyacrylamide gel electrophoresis and stained with Fast Green or periodic acid-Schiff reagents. Radioactivity was measured in gel slices by liquid scintillation spectrometry. This study has shown anomalies of the myelin-associated glycoprotein in the myelin-deficient mutants which could impair the recognition between oligodendrocyte and axon, and consequently involve an arrest in myelinogenesis. These and other results from this laboratory suggest a role of this glycoprotein in the processes of myelination.

Incorporation of (35S) sulfate into chondroitin sulfates by organized cultures of murine peripheral, sympathetic and central nervous tissues.

AbstractOrganized cultures of mouse dorsal root ganglia (PNS), cerebellum (CNS), and sympathetic chain ganglia (ANS) were exposed to feeding media containing radioactive Na235SO4 for 10 day periods beginning either at the onset of myelination or during myelin maintenance. During this period, the used medium was collected at each of three feedings and frozen. Some cultures were frozen and together with the collected medium were analyzed for mucopolysaccharides (MPS). Sister cultures were fixed in ruthenium red‐glutaraldehyde and processed for [35S] radioautography by light microscopy, and cellular localization of MPS by electron microscopy. [35S] MPS were isolated from both cultures and medium (by alkali treatment, proteolytic digestion, TCA treatment, and dialysis, followed by precipitation with cetylpyridinium chloride and ethanol). Isolated MPS were analysed by paper chromatography after digestion with chondroitinase‐ABC and testicular hyarulonidase. Fifty‐five to seventy‐five percent of the total sulfated MPS formed in all types of cultures were chondroitin sulfates (Ch‐S) A, B, and C, ChS‐A, ChS‐B, and ChS‐C are used throughout to indicate chondroitin ‐4‐ sulfate, dermatan sulfate, and chondroitin ‐6‐ sulfate, respectively chondroitin sulfate A accounting for 50 to 60% of the total MPS. PNS and ANS exceeded CNS cultures in total sulfated MPS formed by 10:1.Qualitatively, CNS cultures produced a higher proportion of ChS‐C and a lower proportion of ChS‐B compared to PNS and ANS. Rutheniumred positive material covered all types of cell surfaces, collagen fibers, and the surfaces of enveloped axons; the layers of compact myelin and its underlying axon‐Schwann cell interface showed no such staining, though it appeared regularly in the external mesaxon.

The geometry of peripheral myelin sheaths during their formation and growth in rat sciatic nerves.

In rat sciatic nerves, a small bundle of fibers was identified in which myelin sheaths were absent at birth, appeared within 3 days, and grew rapidly for 2 wk. During this interval, nerves were removed from littermates and were sectioned serially in the transverse plane. Alternating sets of thin and thick sections were used to prepare electron micrograph montages in which single myelinating axons could be identified and traced distally. During the formation of the first spiral turn, the mesaxon's length and configuration varied when it was studied at different levels in the same Schwann cell. The position of the mesaxon's termination shifted while its origin, at the Schwann cell surface, remained relatively constant. Along myelin internodes composed of two to six spiral turns, there were many variations in the number of lamellae and their contour. Near the mesaxon's origin, longitudinal strips of cytoplasm separated the myelin layers. Thicker sheaths were larger in circumference, more circular in transverse sections, and more uniform at different levels. Irregularities were confined to the paranodal region, and separation of lamellae by cytoplasm occurred at Schmidt-Lantermann clefts. Approximate dimensions of the bundle, its largest fibers, and their myelin sheaths were measured and calculated. The myelin membrane's transverse length and area increased exponentially with time; the growth rate increased rapidly during the formation of the first four to six spiral layers and remained relatively constant during the subsequent enlargement of the compact sheath.

Wallerian degeneration in the optic nerve of a reptile: an electron microscopic study.

AbstractNumerous optic nerve fibers persist for a period of up to 20 months following enucleation in reptiles, although it is unlikely that a significant number of efferent fibers are present. After varying survival periods, almost all nerve fibers display distinct morphological changes probably associated with degeneration, but most features previously associated with early stages of degeneration can be seen in long‐survival material except for the early and rapid loss of all non‐myelinated fibers and a honeycomb tubular degeneration associated with the inner and outer myelin layers. Distinct sequential stages of axon and myelin degeneration could not be ascertained despite the slow rate of degeneration in poikilotherms. The varieties of abnormal axon morphology are documented and considered in terms of current light microscopic staining methods.

Theory of the flow of action currents in isolated myelinated nerve fibers. IX.

We continue the presentation of the theory of the isolated fiber.' Effect of Saponin upon the Resting Membrane Potential.-According to reports in the literature,2 3 2 per cent saponin in Ringer's does not depolarize the internodes of isolated fibers until, after long-lasting action (20-40 min), it causes disintegration of the myelin layer. However, when, instead of a condenser-coupled amplifier which cannot detect flows of demarcation current, a d.c.-coupled amplifier is used, it is found that 2 per cent saponin is an exceedingly powerful depolarizing agent; indeed, it is stronger than 114 mM potassium chloride. The depolarization by saponin begins almost instantaneously and increases progressively during a long period of time (no less than 40-60 min). The experiments illustrated by Figures 1-3 belong to a series of 12 in which perfectly consistent results were obtained, for which reason only two experiments will be discussed in which node N, was placed in different positions. In all cases, at the end of the experiment microscopic examination (300 X) failed to detect structural changes in the treated segment, which is in agreement with the fact that in fibers of desheathed nerves kept in 2 per cent saponin for 30 min the myelin layer is stained by osmic acid in apparently the same manner as in untreated nerves. Node N, at the Center of the First Gap and Exposed N2 in the Distal Pool.-The experiment illustrated by Figure 1 is exceptional, since it is the only one in which, with node N, at the center of the first gap, an early downward peak did not appear when the amplifier was placed across the first gap (Fig. 1, 1). In all probability, the stimulation threshold in the central pool was higher than in all the other similar experiments. Nevertheless, the threshold still was low enough (cf. ref. lb) to permit the creation of an active zone in the central pool when a zero-resistance shunt was placed across the first gap (Fig. 1, 2). That the stimulation threshold was relatively high in the central pool is also proved by the fact that with the amplifier in position IV, a deflection was recorded (Fig. 1, 3) having a small downward phase and a large second upward phase (refs. 1 c, e, h). In view of the rapid action of 20 mM xylocaine (Fig. 1, 4-8), it is clear that the active zone responsible for the second upward phase was located in the distal pool. The secondary action of the anesthetic, which also developed rapidly, depressed the state of the isolated fiber. For this reason, with the amplifier across the first gap, an active zone was not created in the central pool and only a trapezoidal action potential was recorded (Fig. 1, 9). With the amplifer across the second gap, in the presence of a zero-

Peculiar particles in macrophage cells of the interstitium of diseased human lungs

Unusual particles in the cytoplasm of the macrophage cells of the interstitium of diseased human lungs are described in this report. The particles are more or less ball shaped and consist of cylindroid tubes in membranous whorls. The size of the particle is about 0.7 μ in diameter. The laminated cylindroid tubes consisting of 3 to 6 myelin layers, are 2800 long and 1000–1400 in diameter. In a cross section of the particle, 20 cylindroid tubes may be seen. The particles may originate as focal accumulations of a finely granulated osmiophilic substance in the macrophages, then the material becomes organized into a particle that consists of the subunit of the cylindroid tubes.

Electron microscopy of focal neuroaxonal lesions produced by beta-beta-iminodipropionitrile (IDPN) in rats. I. The advanced lesions.

An electron microscopic study was made on neuroaxonal lesions in the anterior motoneurons of the lumbar spinal cord in IDPN-treated rats which manifested the permanent “waltzing syndrome” during the stages of axonal balloon formation. The following observations and discussion were made. 1. Within the axonal balloons, there were focal accumulations of usual axoplasmic constituents, viz., neurofibrils, mitochondria, and small vesicles. 2. The balloons, outlined by a thin myelin or axolemmal layer, or axoplasmic contents without definite membrane structure, were often surrounded by edematous spaces filled with granular material. 3. Distal to the balloons, there were segmental accumulations of degenerating mitochondria, vesicles, and also of dense complex bodies within the myelinated portions of axons. 4. No significant alterations were observed in the perikarya of the anterior motoneurons. 5. The present study seems to support a previously held hypothesis of a mechanism of “primary axostasis” as the cause of the lesions as well as of the waltzing syndrome. The intra-axonic dense complex bodies, resembling pigment granules, would provide a plausible explanation for the permanence of the induced syndrome.

The determination of the fourier transform of the myelin layer from a study of swelling phenomena

When the myelin sheath of peripheral nerve is expanded in hypotonic solutions the lipoprotein layers become separated by water layers but remain structurally intact. Consequently the changes in X-ray diffraction intensities which accompany the swelling can be used to deduce the shape of the Fourier transform of the myelin layer. This provides information required for the calculation of the electron density distribution through the layer. The response of the myelin of optic nerve to hypotonic conditions differs from that of sciatic in ways which indicate significant differences in molecular constitution. Studies have also been made of the swelling in hypotonic solutions of frog sciatic nerve already modified by freezing and thawing.

A comparison of the effects of freezing and of treatment with hypertonic solutions on the structure of nerve myelin

When frog peripheral nerve was frozen or immersed in hypertonic solutions, the changes which took place in the low angle X-ray diffraction pattern seemed to reflect a contraction of the lipoprotein structure in some regions of the myelin from an initial periodicity of 171 Å to one of 120 Å. This interpretation was supported by the demonstration of contracted myelin layers in electron micrographs of specimens fixed under hypertonic conditions.

An electron microscope study of degenerative changes in human cutaneous nerve.

A biopsy preparation of human cutaneous nerve from a case diagnosed as a Guillain Barré syndrome has provided extensive electron microscope data relating to nerve degeneration. Fibers in advanced stages of degeneration showed extensive or complete loss of axonal material and the myelin sheaths showed varying degress of fragmentation leading to the formation of independent myelin droplets in which the normal characteristics of the myelin layers were maintained. It is suggested that this fragmentation of the myelin sheath may be due largely to physical stresses which arise when the axonal material is removed.

Electron microscope and x-ray diffraction studies of the effects of dehydrations on the structure of nerve myelin. I. Peripheral nerve.

The dehydration of frog sciatic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. Low magnification electron micrographs of OsO(4)-fixed preparations showed marked tissue shrinkage which could be correlated quantitatively with the loss of water during the preliminary drying. KMnO(4)-fixation appeared to cause a rehydration of the dried tissue. Higher magnification electron micrographs of the OsO(4)-fixed preparations showed a sequence of modifications of the myelin layers which could be correlated with changes in the small-angle x-ray diffraction data which were recorded during drying. An intermediate stage of drying was characterised by a partial collapse of layers and a disappearance of the intraperiod dense line in some regions of the myelin sheath. Continuity between collapsed and non-collapsed layers was maintained throughout the sheath. The fully dried preparation showed two main modifications of the myelin layers. In many regions the layers (principal layers) resembled those of normal preparations, but showed an intensification and frequently a doubling of the intraperiod dense line. In addition, there was a very extensive system of fine (40 A periodicity) dense layers, some of which could be demonstrated to be continuous with the principal layers. In such cases it was observed that two of the fine layers were related to each principal layer. The correlation between diffraction data and electron microscope data is discussed, and some speculations are made concerning the molecular significance of the observations.

Electron microscope and x-ray diffraction studies of the effects of dehydration on the structure of nerve myelin. II. Optic nerve.

The dehydration of rat optic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. A correlation is established between electron micrographs of the myelin sheath and corresponding small-angle x-ray diffraction patterns. The modifications of the optic nerve myelin layers during drying were very similar to those described in more detail for the myelin of frog sciatic nerve. The most striking difference was that the system of fine layers characteristic of the fully dried myelin was much more extensive in the case of the optic nerve, and the layer thickness was significantly greater than the corresponding layer in the frog sciatic nerve preparation. The significance of these correlations is discussed.

The fine anatomy of the optic nerve of anurans--an electron microscope study.

In the optic nerve of Anurans numerous myelinated and unmyelinated axons appear under the electron microscope as compact bundles that are closely bounded by one or several glial cells. In these bundles the unmyelinated fibers (0.15 to 0.6 micro in diameter) are many times more numerous than the myelinated fibers, and are separated from each other, from the bounding glial cells, or from adjacent myelin sheaths, by an extracellular gap that is 90 to 250 A wide. This intercellular space is continuous with the extracellular space in the periphery of the nerve through the numerous mesaxons and cell boundaries which reach the surface. Numerous desmosomes reinforce the attachments of adjacent glial membranes. The myelinated axons do not follow any preferential course and, like the unmyelinated ones, have a sinuous path, continuously shifting their relative position and passing from one bundle to another. At the nodes of Ranvier they behave entirely like unmyelinated axons in their relations to the surrounding cells. At the internodes they lie between the unmyelinated axons without showing an obvious myelogenic connection with the surrounding glial cells. In the absence of connective tissue separating individual myelinated fibers and with each glial cell simultaneously related to many axons, this myelogenic connection is highly distorted by other passing fibers and is very difficult to demonstrate. However, the mode of ending of the myelin layers at the nodes of Ranvier and the spiral disposition of the myelin layers indicate that myelination of these fibers occurs by a process similar to that of peripheral nerves. There are no incisures of Schmidt-Lantermann in the optic myelinated fibers.

Cellular mechanism of myelination in the central nervous system.

A study of myelination with electron microscopy has been carried out on the spinal cord of young rats and cats. In longitudinal and transverse sections the intimate relationship of the growing axons with the oligodendrocytes was observed. Early naked axons appear to be embedded within the cytoplasm and processes of the oligodendrocytes from which they are limited only by the intimately apposed membranes of both elements (axon-oligocytic membrane). In a transverse section several axons are observed to be in a single oligodendrocyte. The process of myelination consists in the laying down, within the cytoplasm of the oligodendrocyte and around the axon, of concentric membranous myelin layers. The first of these layers is deposited at a certain distance (200 to 600 A or more) from the axon-oligocytic membrane. This and all the other subsequently formed membranes have higher electron density and are apparently formed by the coalescence and fusion of vesicles (of 200 to 800 A) and membranes found in large amounts within the cytoplasm of the oligodendrocytes. At an early stage the myelin layers may be discontinuous and some vesicular material may even be trapped among them or between the myelin proper and the axon-oligocytic membrane. Then, when the 8th to 10th layer is deposited, the complete coalescence and alignment of the lamellae leads to the characteristic orderly multilayered organization of the myelin sheath. Myelination in the central nervous system appears to be a process of membrane synthesis within the cytoplasm of the oligodendrocyte and not a result of the wrapping of the plasma membranes as postulated in Geren's hypothesis for the peripheral nerve fibers. The possible participation of Schwann cell cytoplasm in peripheral myelination is now being investigated.

Effects of ionic strength of immersion medium on the structure of peripheral nerve myelin.

A study of the effects of hypertonic solutions on the structure of peripheral nerve myelin reveals an expansion rather than a contraction of the layer spacing. This suggests the absence of "free" water between the myelin layers. Hypotonic solutions bring about a change in radial repeat period from 171 A to 250 to 270 A. These findings are of significance in relation to the structure of myelin.

Outflow from cut ends of nerve fibres

When the axoplasm of isolated nerve fibres of frog is fluid, the content emerges slowly from the cut ends of the fibres, forming a spherical droplet of axoplasm invested by a layer of myelin. The volume of the discharged axoplasm is proportional to the square root of the time. The terminal portion of the fibre narrows during the outflow. The reduction of diameter is most marked at the cross-section and fades gradually away with the increasing distance from the cut end. When the outflowing drop reaches a definite magnitude, the flow is arrested and the volume of the drop becomes for a time stationary. The stationary period is ended when the myelin membrane coating the drop bursts. The outflow is resumed immediately afterwards. The mechanism of the outflow from cut nerve fibres is discussed.

Structure of the motor endplate

TN his comprehensive review on innerva| tion of skeletal muscle, Tiegs I quotes I statement made by Kuffier ~ that the end plate has become, in recent years, a physiological entity which at present cannot be strictly identified with structure of histologists. One reason: for this hiatus in our knowledge is disagreement which exists among histologists concerning embryologic derivation and limiting boundaries of some of structures which comprise endplate. The controversial points are discussed in detail in Tiegs' review; present description is drawn chiefly from pioneer work of Kiihne ~ and more recent studies of Couteaux 4,5 and Young 6 and their associates. Centrally to its point of contact with muscle fiber, motor axon is ensheathed in a layer of myelin which is closely invested by Schwann cells, The latter are surrounded by a continuous sheath called neurilemma external to which is sheath of Henle, a continuation of perineural sheath surrounding entire bundle of nerve fibers. Near endplate region myelin sheath is lost, and sheath of Henle becomes continuous with outer sarcolemma, an extraneous sheath derived from endomysium. According to Couteaux, 4 at endplate axon retains a surrounding sheath, teloglia, derived from sheath of Schwann, and remains external to true sarcolemma. The latter is modified, beneath axon with its investing teloglia, as pallisade-like subneural apparatus. Whether neurilemma or a derivative is retained is less certain. Surrounding subneural apparatus is a fairly well circumscribed region of granular sarcoplasm which is rich in nuclei. The entire motor endplate occupies a third or less of circumference of muscle fiber. Couteaux has focussed attention on subneural apparatus as probable site of both

Die Signalübermittlung im Nerven

PROF. v. MURALT and his collaborators in Bern have taken up and developed the technique for the isolation of single frog‘s nerve fibres invented by the Japanese, Kato and Tasaki. As a result of optical and biochemical investigations of great technical ingenuity he has developed theories about the mechanism of conduction in nerve which might be described as unorthodox. The basic unit of conduction is supposed to be the internode of medullated nerve fibres, and v. Muralt believes that each is separated from the next internode by a transverse membrane at the node of Ranvier. Unfortunately his pictures do not provide decisive evidence of the existence of transverse membranes crossing the entire axon at the node. The optical appearances which they show are well known to histologists and are usually considered as representing the abrupt end of the myelin layer. His belief in the existence of this membrane was strengthened by the observation that in dying fibres it was sometimes seen to be blown out in the direction of one segment or the other. But the figure demonstrating this effect is not convincing and would be taken by most to show a modified portion of myelin rather than a membrane across the axon. However, the exact details of the node of Ranvier and the nature of the structure called by Cajal the cementing disk still remain very obscure, and v. Muralt‘s provocative study will certainly stimulate further investigation. Die Signalubermittlung im Nerven By Prof. Alexander v. Muralt. (Lehrbucher und Monographien aus dem Gebiete der Exakten Wissenschaften, Band 14: Reihe der experimentellen Biologie, Band 3.) Pp. 354. (Basel: Verlag Birk-hauser, n.d.) 38.50 Schw. francs.