Structural alterations in nerve fibers produced by hypotonic and hypertonic solutions

In sections of KMnO(4)-fixed, developing mouse sciatic nerves, the central gap of mesaxons in myelinating fibers is normally closed with close apposition of the outside approximately 20 A dense strata of the two approximately 75 A Schwann cell membranes. The two combined outside strata make the intraperiod line bisecting each myelin lamella. The approximately 150 A mesaxon is elaborated spirally around the axon in either a right hand or left hand spiral, and its inside (cytoplasmic) approximately 20 A strata in apposition form the major dense lines of myelin. In hypotonic solutions the lamellae of adult frog sciatic myelinated fibers split apart along the outside membrane strata apposed at the intraperiod line throughout the spiral. Under similar conditions the inside (cytoplasmic) strata of the membranes, in apposition at the major dense lines, do not separate. The approximately 150 A membranous structure resulting from this is called an "internal compound membrane." The double membranes of normal and control frog sciatic unmyelinated fibers have a central gap approximately 100 to 150 A wide. After soaking in 4 to 10 times normal strength Ringer solution or 10 N sucrose-Ringer solution, this gap closes and a membranous structure approximately 150 A wide resembling developing mouse mesaxons results. This is designated by the term "external compound membrane." The latter membranes resemble internal compound membranes, but their central dense zones, each consisting of two apposed outside membrane strata, are less dense.     

FURTHER OBSERVATIONS ON THE STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

Direct evidence has been presented to confirm the existence of a spiral in the myelin sheaths of the central nervous system. An account of some of the variations in structure of central myelin sheaths has been given and it has been shown that the radial component of myelin sheaths has the form of a series of rod-like thickenings of the intraperiod line. These thickenings extend along the intraperiod line in a direction parallel to the length of the axon. The relative position of the internal mesaxon and external tongue of cytoplasm has been determined in a number of transverse sections of sheaths from the optic nerves of adult mice, adult rats, and young rats. In about 75 per cent of the mature sheaths examined, these two structures were found within the same quadrant of the sheath, so that the cytoplasm of the external tongue process tends to lie directly outside that associated with the internal mesaxon. The frequency with which the internal mesaxon and external tongue lie within the same quadrant of the sheath increases both with the age of the animal and with the number of lamellae present within a sheath. The possible significance of these findings is discussed.     

THE FORMATION AND STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

The development and structure of myelin sheaths have been studied in the optic nerves of rats and of Xenopus laevis tadpoles. Both potassium permanganate- and osmium-fixed material was examined with the electron microscope. In the first stage of myelinogenesis the nerve fibre is surrounded by a cell process which envelops it and forms a mesaxon. The mesaxon then elongates into a loose spiral from which the cytoplasm is later excluded, so that compact myelin is formed. This process is similar to myelinogenesis in the peripheral nervous system, although in central fibres the cytoplasm on the outside of the myelin is confined in a tongue-like process to a fraction of the circumference, leaving the remainder of the sheath uncovered, so that contacts are possible between adjacent myelin sheaths. The structure of nodes in the central nervous system has been described and it is suggested that the oligodendrocytes may be the myelin-forming cells.     

ELECTRON MICROSCOPIC OBSERVATIONS OF RAT AND MOUSE CEREBELLUM IN TISSUE CULTURE

Closely ordered stages of myelin formation in cultures of newborn rat and mouse cerebellum, selected by direct light microscopy, were studied with the electron microscope. Electron micrographs of these cultures reveal the presence of neurons, axons, neuroglia, microglia, and ependymal cells. The appearance of the neuron is identical to that previously described in vivo. The neuroglial cell has long, branching processes, and its cytoplasm is characterized by packets of long, narrow fibrils. During myelin formation, a glial cell process surrounds the axon. This process may form an internal mesaxon and may spiral for several turns around the axon. Other glial cell processes may interdigitate with or overlay the innermost process to contribute to the multilamellated structure. The glial processes flatten and the cytoplasmic surfaces of the cell membrane come into contact to form the lamellae of the myelin sheath. These adhesions may be temporarily incomplete as evidenced by sequestered islands of glial cytoplasm among the myelin lamellae. Ultimately, a compact, apparently spiral, myelin sheath is formed. These findings are discussed in relation to in vivo central myelin formation.     

ULTRASTRUCTURAL STUDY OF REMYELINATION IN AN EXPERIMENTAL LESION IN ADULT CAT SPINAL CORD

This report presents ultrastructural observations on the cytological events that attend myelin formation occurring in the wake of demyelination in adult cat spinal cord. Lesions were induced in subpial cord by cerebrospinal fluid (c.s.f.) exchange (1, 2). Tissue from eleven cats at nine intervals from 19 to 460 days was fixed in situ by replacing c.s.f. with buffered OsO₄ and embedded in Araldite. After demyelination, axons are embraced by sheet-like glial processes. An occasional myelin sheath is first seen at 19 days; by 64 days, all axons are at least thinly myelinated. The cytoplasm of the myelin-forming cells, unlike that of either oligodendrocyte or fibrous astrocyte in normal cord, is dense with closely packed organelles and fine fibrils. Many of the myelinogenic cells become scarring astrocytes and at 460 days the lesion teems with their fibril-filled processes. Oligodendrocytes appear in the lesion after remyelination is under way. Phagocytes disappear gradually. A myelin sheath is formed by spiral wrapping of a sheet-like glial process around an axon. Where the first turn of the spiral is completed, a mesaxon is formed. As cytoplasm is lost from the process, the plasma membrane comes together along its outer and cytoplasmic surfaces to form compact myelin. Only a small amount of cytoplasm is retained; it is confined to the paramesaxonal region and, on the sheath exterior, to a longitudinal ridge which appears in profile as a small loop. This outer loop has the same rotational orientation as the inner mesaxon. These vestiges of spiral membrane wrapping are also found in normal adult and new-born cat cord. Nodes are present in all stages of remyelination and in normal adult cat and kitten cord. These observations suggest that myelin is reformed in the lesion in the same way it is first formed during normal development. The mechanism of myelin formation is basically similar to that proposed for peripheral nerve and amphibian and mammalian optic nerve; it does not agree with present views on the mechanism of myelinogenesis in mammalian brain and cord. This is the first demonstration of remyelination in adult mammalian central nervous tissue.     

Accessibility of Galactosyl Ceramides to Probe Reagents in Central Nervous System Myelin

The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid- and nonhydroxy fatty acid-containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20°C and electron micrographs of the periodate-treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.     

Morphological and morphometric studies of early myelination in the cervical dorsal funiculus of the newborn mouse spinal cord

The early myelination of the dorsal funiculus at the level of the 4th cervical spinal cord was ultrastructurally studied in the one-day-old mouse. It was found that the fibers were mainly unmyelinated. However, some early myelinated fibers were scattered among unmyelinated fibers. In the initial stage of the myelination, the axon was partially contracted by a piece of cytoplasmic process of the oligodendroglial cell. The two lips of the oligodendroglial process then extended and converged, enwrapping the axon completely and forming the first contact point. With the anchorage of that contact point, the two lips of the process became elongated and enfolded by each other, and produced the internal and external tongues of the future myelin sheath. More contact points were formed at a regular interval by the regional fusion of the two external surface layers of the opposed cytoplasmic membranes of adjacent tongue processes. With the advanced bidirectional spiralization of the two tongue processes, many contact points were found between the adjacent lamellae of the concentrically arranged oligodendroglial process; simultaneously, the cleft between the neighboring contact points disappeared and formed the initial sites of the intraperiod line. During the early myelination, one single axon ensheathed concentrically by two different oligodendroglial processes as well as several axons enwrapped by a continuous spiral myelin sheath of one oligodendroglial cell were frequently observed. The cross-sectional areas of unmyelinated axons varied from 0.01 to 0.2 micron 2, with a median of 0.07 micron 2; whereas, that of promyelinated axons ranged from 0.09 to 1.4 micron 2, with a median at 0.61 micron 2. These data support the suggestion that the axon calibre is a critical factor for the initiation of central myelination.     

Peculiarities of myelin formation in the spinal roots of the rabbit]

The axon sheath formation in the ventral and dorsal spinal roots of newborn rabbits is discussed. The mesaxon grows at a greater rate than the outer plasmalemma of the Schwann cell, thereby giving rise to folds in the mesaxon. The compact myelin spiral is formed by the apposition of 2-3 lamellae.     

Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin

The myelin-associated glycoprotein (MAG) is an integral membrane protein (congruent to 100,000 mol wt) which is a minor component of purified peripheral nervus system (PNS) myelin. In the present study, MAG was localized immunocytochemically in 1-micrometer thick Epon sections of 7-d and adult rat peripheral nerves, and its localization was compared to that of the major structural protein (Po) of PNS myelin. To determine more precisely the localization of MAG, immunostained areas in 1 micrometer sections were traced on electron micrographs of identical areas from adjacently cut thin sections.l MAG was localized in periaxonal membranes. Schmidt-Lantermann incisures, paranodal membranes, and the outer mesaxon of PNS myelin sheaths. Compact regions of PNS myelin did not react with MAG antiserum. The results demonstrate MAG's presence in "'semi-compact" Schwann cell or myelin membranes that have a gap of 12-14 nm between extracellular leaflets and a spacing of 5 nm or more between cytoplasmic leaflets. In compact regions of the myelin sheath which do not contain MAG, the cytoplasmic leaflets are "fused" and form the major dense line, whereas the extracellular leaflets are separated by a 2.0 nm gap appearing as paired minor dense lines. Thus, it is proposed that MAG plays a role in maintaining the periaxonal space, Schmidt-Lantermann incisures, paranodal myelin loops, and outer mesaxon by preventing "complete" compaction of Schwann cell and myelin membranes. The presence of MAG in these locations also suggests that MAG may serve a function in regulating myelination in the PNS.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION 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 OsO₄-fixed preparations showed marked tissue shrinkage which could be correlated quantitatively with the loss of water during the preliminary drying. KMnO₄-fixation appeared to cause a rehydration of the dried tissue. Higher magnification electron micrographs of the OsO₄-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.     

Ballooning of myelin sheaths in normally aged macaques

In aged animal brains, a variety of holes are formed in the neuropil. One type of hole, here designated as the myelin balloon, is an abnormality of the myelin sheath and is found in a number of diverse sites in the brain. Profiles of myelin balloons display rather smoothly rounded peripheral contours and typically range up to 10 μm in diameter, although exceptionally large examples may be twice this size. The balloons are bounded by lamellae of myelin, and to accommodate the contents of the balloon, the myelin sheath becomes split at the intraperiod line. Since the intraperiod line is formed by the apposition of the outer faces of the myelin-forming plasma membrane, the contents of the myelin balloons are, in effect, in continuity with the extracellular space, and it is suggested that the contents of the balloons are fluid, with the fluid exerting an outward pressure on the walls of the balloons to produce their spherical shapes. Myelin balloons are not only produced during aging but also occur in a number of genetic strains of mice and in a number of human disease states. They thus represent a non-specific, though distinctive and common, alteration of the myelin sheath and are a reflection of the fact that under a variety of conditions, including normal aging, oligodendrocytes are unable to maintain the integrity of their sheaths.     

Does a cytoplasmic bridge connect the CNS axon with the inner loop of myelin?

Sarah Luse (1959) reported over 30 years ago on the presence of a bridge connecting the axon to the myelin sheath in the central nervous system (CNS). This notion has not been accepted in the literature. Wolman (1992) found that the progress of demyelination in some viral diseases affecting the CNS fits the concept of Luse, as the process occurred primarily along the major dense line of myelin, which is in continuity with the cytoplasm of the oligodendroglial cell. Injection of Lucifer yellow (LY) and horseradish peroxidase (HRP) into the vitreous of guinea pigs, with and without iontophoresis, resulted in labeling of the nerve axons and myelin. Labeling of myelin by HRP occurred along the major dense line which indicated that a transient or permanent cytoplasmic bridge connects axons and myelin in the optic nerve.     

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.     

Ultrastructural sequence of myelin breakdown during Wallerian degeneration in the rat optic nerve

Summary Adult albino rats were subjected to unilateral surgical removal of the eyeball. After survival times of 7–140 days, the numerical response of the neuroglial cells, and the progressive disintegration of the myelin sheaths in the optic nerves, were studied qualitatively and quantitatively in electron-microscopic montages. The distribution density of microglia and astroglia in degenerating optic nerve increased to peaks after 35 and 56 days respectively, whereas, the oligodendroglia gradually decreased. During the early stage of degeneration, microglial cells appeared and invaded the sheath at the intraperiod line, peeling off the outer lamellae, which were then engulfed by phagocytosis. Within the microglia, myelin sheath fragments were surrounded by a membrane curled to form a myelin ring. In the intermediate stage of degeneration, the paired electrondense lines of the ring, made up of myelin basic protein, decomposed and formed a homogenous or heterogenous osmiophilic layered structure, the myelin body, which, in the final stages, disintegrated and transformed into globoid lipid droplets and needle shaped cholesterol crystals.     

Cytochemical localization of ATPase in axon-myelin-Schwann cell complex type

ATPase activity was studied in the structures of axon-myelin-Schwann cell complex of sciatic nerves of rabbits of pre-and postnatal development. Positive reaction was observed on the plasma membrane, mitochondria and endoplasmic reticulum of Schwann cells, on the intraperiod lines of the compact myelin, in the split myelin lamellae in the paranodal regions and Schmidt-Lanterman clefts, in segment of outermost lamellae split off from the interparanodal myelin, in the mesaxons, in the loose myelin lamellae in the earlier stages of myelinization, on the axolemma (periaxonal space) and axoplasm. The ATPase activity on the Schwannian plasmalemma, axolemma and myelin sheath surface was found to be heterogeneously distributed. An accumulated of reaction deposits at the origin of the outer mesaxon, at the axoglial contacts as well as at the terminal part of the myelin sheath was respectively observed. Alterations of the enzyme activity distribution in axon-myelin-Schwann cell complex during rabbit's development were found to be associated with the growing myelin sheath and its node-paranode. Using controls with ouabain an attempt was made the possibilities of Wachstein and Meisel's method to be shown and the place of alpha+ form of Na+, K+-ATPase in the axon-myelin-Schwann cell Complex to be establish.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Some Observations on the Fine Structure of the Giant Nerve Fibers of the Earthworm, Eisenia foetida

Sectioned dorsal giant fibers of the earthworm Eisenia foetida have been studied with the electron microscope. The giant axon is surrounded by a Schwannian sheath in which the lamellae are arranged spirally. They can be traced from the outer surface of the Schwann cell to the axon-Schwann membranes. Irregularities in the spiral arrangement are frequently observed. Desmosome-like attachment areas occur on the giant fiber nerve sheath. These structures appear to be arranged bilaterally in columns which are oriented slightly obliquely to the long axis of the giant fiber and aligned linearly from the axon to the periphery of the sheath. At these sites they bind together apposing portions of Schwann cell membrane comprising the sheath. Longitudinal or oblique sections of the nerve sheath attachment areas are reminiscent of the Schmidt-Lantermann clefts of vertebrate peripheral nerve. Septa of the giant fibers have been examined. They are symmetrical or non-polarized and consist of the two plasma membranes of adjacent nerve units. Characteristic vesicular and tubular structures are associated with both cytoplasmic surfaces of these septa.     

Centrifugal decrement in diameter of myelinated afferent fibres innervating frog taste organ

1. Regional changes in the diameter of single myelinated afferent nerve fibres innervating the taste disc of the fungiform papillae on the bullfrog tongue were investigated morphologically and functionally. 2. The diameter of myelinated afferents in the medial lingual branch of the glossopharyngeal nerve averaged 8.4 microns at the proximal end of the tongue and gradually decreased at the rate of 0.8 micron/cm length of the fibres as they ran in the apical direction of the tongue. 3. The conduction velocity of single myelinated afferent fibres within the tongue decreased gradually as they ran peripherally. 4. Electrophysiological inspection of neural connections between the fungiform papillae suggests that a gradual centrifugal decrease in the diameter of a single myelinated afferent fibre is not due to multiple bifurcations of the fibre at various sites within the tongue, but due to a natural gradual decrease in the thickness of the myelin sheath and the diameter of axon.