The Ultrastructure of Schmidt-Lanterman Clefts and Related Shearing Defects of the Myelin Sheath

Schmidt-Lanterman clefts in frog sciatic nerves have been studied in thin sections by electron microscopy utilizing permanganate fixation and araldite embedding. It is shown that they are shearing defects in myelin in which the lamellae are separated widely at the major dense lines. Each lamella consisting of two apposed Schwann cell unit membranes ~ 75 A across traverses the cleft intact. The unit membranes composing each lamella sometimes are slightly (~ 50 to 100 A) separated in the clefts. The layers between the lamellae contain membranous structures which may be components of the endoplasmic reticulum. These layers are continuous with the outer layer of Schwann cytoplasm and the thin and inconstant cytoplasmic layer next to the axon (Mauthner's sheath). Each of these layers in perfect clefts constitutes a long helical pathway through the myelin from the axon. One of these is connected with Schwann cytoplasm and the other directly with the outside. A type of cross-sectional shearing defect, not hitherto recognized, is described and shown to be a kind of Schmidt-Lanterman cleft. Incomplete clefts are seen and interpreted as representing stages in a dynamic process whereby the myelin lamellae may be constantly separating and coming together again in life.     

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.     

神经退变和再生的构筑变化

将夹伤的大鼠坐骨神经分离成单根纤维,观察98d内轴突和许旺细胞的构筑变化过程发现,损伤既使轴浆转运阻断、积累的细胞器退变,也使髓鞘板层,特别是斯兰氏切迹撕裂、变形或侵入轴突。轴突或髓鞘虽可各呈单一的退变,但以两者并存多见。伤后1d即出现富含微管的再生芽,它被增殖的许旺细胞突起及其基底膜包绕,并逐步发育成熟。根据再生的特征性构筑变化,提出了再生芽、无髓和有髓纤维、斯兰氏切迹、朗氏结与神经小束的初见、发育和成熟高峰期的时间顺序。无髓纤维的发育成熟早于有髓纤维。     

Schmidt-Lanterman clefts: a morphometric study in human sural nerve

In the human sural nerve, large myelinated fibers contained 35 Schmidt-Lanterman (SL) clefts per mm, and small myelinated fibers contained only eight SL clefts per mm. The incidence of SL clefts is linearly related to myelin thickness. The SL clefts extended over 13 micron in large and over 9 micron in small fibers, the total extent of the SL region amounting to nearly 50% of internodal length in large and to 6% in small fibers. In the SL region, the fiber diameter was 6% larger than outside this region, and the axon was 17% smaller in large and 28% smaller in small fibers. The paranodal-nodal region occupied less than 2% of internodal length in large fibers and 6.5% in small fibers; in the nodal region the axon diameter was reduced by 40-50%.     

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.     

Morphology of Schmidt-Lanterman incisures of human shoulder plexus nerves in ontogenesis

The Schmidt-Lanterman incisures are formed in the phase of "loose" myelin at 15-17 weeks of prenatal development with the formation of nodes of Ranvier. The formation of mature Schmidt-Lanterman incisure is completed in the second foetis period. Schmidt-Lanterman incisures distribution in nerve fibres of young men depends on fibre diameter. In the nerves of elderly people the distribution of the incisures in the internode doesn't depend on a fibre diameter. In the nerves of the elderly men the number of incisures is twice as much as that in the young people. The incisural ultrastructure in human nerve is similar to its structure in other mammals.     

Zur Mechanik der Markballenbildung bei der Wallerschen Degeneration

Summary Wallerian degeneration was studied in frog single fibers. A new preparation for microscopic observation of peripheral motor fibers in living Rana esculenta was used. The authors studied proximally transected groups of fibers using the polarization technique.The results confirmed that the axon destruction preceds the breakdown of the myelin sheath, and the Schmidt-Lanterman incisures play an important role in the formation of ovoids. Details of the ovoid formation are demonstrated in serial photographs. It is suggested that the incisures of Schmidt-Lanterman are pathways for electrolyte and water transport in Wallerian degeneration.     

Immunocytochemical localization of P0 protein in Golgi complex membranes and myelin of developing rat Schwann cells

P0 protein, the dominant protein in peripheral nervous system myelin, was studied immunocytochemically in both developing and mature Schwann cells. Trigeminal and sciatic nerves from newborn, 7-d, and adult rats were processed for transmission electron microscopy. Alternating 1- micrometer-thick Epon sections were stained with paraphenylenediamine (PD) or with P0 antiserum according to the peroxidase-antiperoxidase method. To localize P0 in Schwann cell cytoplasm and myelin membranes, the distribution of immunostaining observed in 1-micrometer sections was mapped on electron micrographs of identical areas found in adjacent thin sections. The first P0 staining was observed around axons and/or in cytoplasm of Schwann cells that had established a 1:1 relationship with axons. In newborn nerves, staining of newly formed myelin sheaths was detected more readily with P0 antiserum than with PD. Myelin sheaths with as few as three lamellae could be identified with the light microscope. Very thin sheaths often stained less intensely and part of their circumference frequently was unstained. Schmidt-Lanterman clefts found in more mature sheaths also were unstained. As myelination progressed, intensely stained myelin rings became much more numerous and, in adult nerves, all sheaths were intensely and uniformly stained. Particulate P0 staining also was observed in juxtanuclear areas of Schwann cell cytoplasm. It was most prominent during development, then decreased, but still was detected in adult nerves. The cytoplasmic areas stained by P0 antiserum were rich in Golgi complex membranes.     

Rafts in adult peripheral nerve myelin contain major structural myelin proteins and myelin and lymphocyte protein (MAL) and CD59 as specific markers

The myelin and lymphocyte protein (MAL) proteolipid is localized in central and peripheral compact myelin membranes, as well as in apical membranes of particular polarized cells. In this study, we addressed the question whether MAL and other peripheral myelin proteins are sorted and targeted to myelin membranes using mechanisms similar to those observed in polarized epithelial cells. To investigate the presence of raft-mediated sorting pathways in Schwann cells, we have isolated and analysed their composition in myelin membranes. Here, we show that rafts are present in adult human and rat peripheral compact myelin membranes and contain MAL, the GPI-anchored protein CD59, and substantial amounts of the PMP22 and P0. Colocalization studies show that CD59, and MAL have an almost identical expression pattern within compact myelin. Moreover, immuno-electron microscopy revealed that MAL, besides its localization in compact myelin, is also localized to Schmidt-Lanterman incisures. Taken together, our results demonstrate the presence of detergent-insoluble glycolipid-enriched complexes (DIGs) in different compartments of myelin membranes and indicate an important role for DIG-mediated transport mechanisms in the maintenance of the adult myelin sheath.     

Ultrastructural distribution of Ca++ within neurons

Summary We used the oxalate-pyroantimonate technique to determine the ultrastructural distribution of Ca⁺⁺ in neurons of the rat sciatic nerve. The content of the precipitate was confirmed by X-ray microanalysis and appropriate controls. In the cell bodies of the dorsal root ganglia, Ca⁺⁺ precipitate was found in the Golgi, mitochondria, multivesicular bodies and large vesicles of the cytoplasm but not in lysosomes, and was prominently absent from regions of rough endoplasmic reticulum and ribosomes. It was seen in the nucleus but not in the nuclear bodies or nucleolus.Within the axon itself, Ca⁺⁺ precipitate was also found sequestered in mitochondria and smooth endoplasmic reticulum. In addition Ca⁺⁺ precipitate found diffusely throughout the axoplasm exhibited a discrete and heterogeneous distribution. In myelinated fibers the amount of precipitate decreased predictably in the axoplasm beneath the Schmidt-Lanterman clefts and in the paranodal regions at the nodes of Ranvier. This correlated with the presence of dense precipitate in the Schmidt-Lanterman clefts them-selves and in the paranodal loops of myelin.Intracytoplasmic ionic Ca⁺⁺ is maintained at 10^–7 M by balanced processes of influx, sequestration and extrusion. The irregular distribution of Ca⁺⁺ precipitate in the axoplasm of myelinated fibers suggests that there may be specific regions of preferential efflux across the axolemma.     

The fine structure of myelinated nerve cell bodies in the bulbus olfactorius of man

Summary In the bulbus olfactorius of man numerous myelinated nerve cell bodies occur in the stratum plexiforme internum and stratum granulosum internum. In many respects they resemble the neighbouring granule cells: small chromatin clumps border on more than half of the circumference of the nucleus, the thin cytoplasmic rim contains abundant polysomes and sometimes pigment complexes with numerous light vacuoles, the cells often show a process which extends up to the stratum glomerulosum, the perikarya are devoid of synaptic contacts whereas the proximal segment of the peripheral processes display rare contacts. The myelin sheath varies in thickness, consisting of 2 to 24 lamellae with distances between the major dense lines ranging from 9.3 to 11.3 nm. The myelin sheath may enclose the cell body completely or partially and accompany the proximal segment of the process arising from the perikaryon. On partially enveloped perikarya, the myelin lamellae end in formations like those of the node of Ranvier, though often less regularly. Within the compact myelin sheath all of its lamellae may be distended for a short distance by glial cytoplasm as in the Schmidt-Lanterman incisures of peripheral nerve fibres. Adjacent to the outermost myelin lamella myelinated axons and cell bodies, tentatively identified as oligodendrocytes, as well as granule cells may be closely joined.Supported by the Deutsche Forschungsgemeinschaft (Br. 634/1)     

Myelinating Schwann cells determine the internodal localization of Kv1.1, Kv1.2, Kvβ2, and Caspr

We examined the localization of Caspr and the K⁺ channels Kv1.1 and Kv1.2, all of which are intrinsic membrane proteins of myelinated axons in the PNS. Caspr is localized to the paranode; Kv1.1, Kv1.2 and their β2 subunit are localized to the juxtaparanode. Throughout the internodal region, a strand of Caspr staining is flanked by a double strand of Kv1.1/Kv1.2/Kvβ2 staining. This tripartite strand apposes the inner mesaxon of the myelin sheath, and forms a circumferential ring that apposes the innermost aspect of Schmidt-Lanterman incisures. The localization of Caspr and Kv1.2 are not disrupted in mice with null mutations of the myelin associated glycoprotein, connexin32, or Kv1.1 genes. At all of these locations, Caspr and Kv1.1/Kv1.2/Kvβ2 define distinct but interrelated domains of the axonal membrane that appear to be organized by the myelin sheath.     

ELECTRON MICROSCOPY OF THE PACINIAN CORPUSCLE

The Pacinian corpuscle has a framework of cytoplasmic lamellae arranged concentrically in the outer zone, and bilaterally in the core. Between these is an intermediate growth zone. The inner core shows an unexpected complexity in that its component lamellae are arranged in two symmetrical groups of nested cytoplasmic sheets. Longitudinal tissue spaces form clefts separating the two groups. The perikarya of the core lamellae lie in or near the intermediate growth zone, and send arms into the clefts. The arms then branch and terminate as lamellae which interdigitate with those of neighboring cells. The single nerve fiber loses its myelin sheath just before it reaches the inner core but retains its Schwann cell cytoplasmic covering for a short additional distance. The Schwann sheath is not continuous with the lamellae of the inner core. Inside the core the fiber contains a striking circumferential palisade of radially disposed mitochondria. The fiber does not arborize. Vascular capillaries penetrate the hilar region of the corpuscle only as far as the myelinated sheath of the nerve, and they have not been seen elsewhere in the corpuscle. There is direct continuity between the clefts of the core and tissue spaces in the vicinity of the capillaries. It is likely that this provides a route whereby metabolites reach the active nerve ending, as well as the cells of the growth zone. The outer zone consists of at least 30 flattened concentric cytoplasmic lamellae separated from one another by relatively wide fluid-filled spaces. Collagenous fibrils are present, particularly on the outer surface of lamellae, and tend to be oriented circularly. The girdle of proliferating cells constituting the growth zone, which is prominent in corpuscles from young animals, is the layer from which the outer lamellae are derived. Osmotic forces probably elevate the lamellae, and maintain turgor pressure.     

Remyelination in the chicken sciatic nerve

Remyelination in the chicken sciatic nerve occurring after the injection of diphtheria toxin was studied. The rates of fast axonal transport and conduction velocities were measured sixty days after the injection of the toxin. Fast axonal transport rates were found to have returned to normal in the remyelinated nerves, but conduction velocity was markedly reduced even though the birds appeared to walk normally. The remyelinated nerve fibres had on histological examination relatively thin myelin sheaths. Of greater interest was the number of Schmidt-Lanterman clefts observed in both the control and remyelinated nerves when viewed in the electron microscope.     

Quantitative analysis of postnatal myelin sheath development in the dorsal funiculus of rat

Postnatal development of myelin sheaths in the dorsal funiculus of rats has been studied qualitatively by light microscopy and morphometry. A distinction is made between Goll's tract and Burdach's tract, and, furthermore, inside Goll's tract the cervical, thoracic and lumbar areas are compared. The induction of myelinization is influenced by a critical range of axon thicknesses. Between the 15th and 20th days of postnatal maturation there is a minimal growth reaction in the dorsal funiculus, but after the 20th day up to the 120th day extensive growth of the myelin sheath can be seen. Burdach's tract shows earlier and faster development of the myelin sheath than Goll's tract cervically, which leads to the conclusion that epicritical sensitivity matures earlier in the upper extremity. Concerning Goll's tract in the lumbar area of the dorsal funiculus, faster maturation than in the thoracic and cervical areas can be seen.     

ELECTRON MICROSCOPE STUDIES OF THE FORMATION OF NODES OF RANVIER IN MOUSE SCIATIC NERVES

Observations with the electron microscope of longitudinal sections of the sciatic nerves of infant mice during the period of early myelin formation are described. These observations are interpreted in relation to previous studies of transverse sections, and a general picture of the formation of an internodal length of the myelin sheath in three dimensions is formulated. In general, an internodal length of myelin sheath is attained by the spiral wrapping of the infolded Schwann cell surface; the increase in length of the internode during maturation is at least partially explained by the increased length of axon covered by the overlapping of successive layers during the wrapping of the infolded Schwann cell surface; and the nodes of Ranvier refer to the structure complex at the junctions of adjacent non-syncytial Schwann cells. The fact that the mode of formation of myelin brings each of its layers into intimate contact with the axon surface at the nodes is emphasized because of the possible functional significance of this arrangement. The manner of origin of Schmidt-Lantermann clefts remains obscure. Certain isolated observations provide evidence for the possibility that occasional internodes of myelin may form from several small segments of myelin within a single Schwann cell.     

Myelin Organization in the Nodal,Paranodal, and Juxtaparanodal Regions Revealed by Scanning X-Ray Microdiffraction

X-ray diffraction has provided extensive information about the arrangement of lipids and proteins in multilamellar myelin. This information has been limited to the abundant inter-nodal regions of the sheath because these regions dominate the scattering when x-ray beams of 100 µm diameter or more are used. Here, we used a 1 µm beam, raster-scanned across a single nerve fiber, to obtain detailed information about the molecular architecture in the nodal, paranodal, and juxtaparanodal regions. Orientation of the lamellar membrane stacks and membrane periodicity varied spatially. In the juxtaparanode-internode, 198–202 Å-period membrane arrays oriented normal to the nerve fiber axis predominated, whereas in the paranode-node, 205–208 Å-period arrays oriented along the fiber direction predominated. In parts of the sheath distal to the node, multiple sets of lamellar reflections were observed at angles to one another, suggesting that the myelin multilayers are deformed at the Schmidt-Lanterman incisures. The calculated electron density of myelin in the different regions exhibited membrane bilayer profiles with varied electron densities at the polar head groups, likely due to different amounts of major myelin proteins (P0 glycoprotein and myelin basic protein). Scattering from the center of the nerve fibers, where the x-rays are incident en face (perpendicular) to the membrane planes, provided information about the lateral distribution of protein. By underscoring the heterogeneity of membrane packing, microdiffraction analysis suggests a powerful new strategy for understanding the underlying molecular foundation of a broad spectrum of myelinopathies dependent on local specializations of myelin structure in both the PNS and CNS.     

Monosialoganglioside (GM1) immunofluorescence in rat spinal roots studied with a monoclonal antibody

Gangliosides are characteristic glycolipid components of plasma cell membranes, especially enriched in the CNS and PNS. In some diseases involving the PNS, in particular motor neuropathies associated with conduction block, IgM autoantibodies against ganglioside GM1 have been implicated as a pathogenic factor. In order to study the GM1 distribution in peripheral nerves we have investigated its in situ localization using a new anti-GM1 monoclonal antibody, GM1:1. Immunization and production of the monoclonal antibody was made by common protocols and binding specificity was investigated by using structurally related glycolipids and modified GM1-molecules. The result showed that an α2–3 bound sialic acid together with a terminal galactose moiety were essential for GM1:1 binding. In situ localization of GM1 in rat dorsal and ventral spinal roots was investigated by conventional immunomicroscopy. GM1 immunoreactivity was the same in both roots and appeared like a finely granular, in places confluent, material confined to Schmidt-Lanterman’s incisures, to myelin sheath paranodal end segments and to some extent to the abaxonal Schwann cell cytoplasm; all of these structures are likely to be the target for GM1 antibodies in peripheral neuropathies. Nodal gaps and fibre contours showed a weak non-specific fluorescence. The localization of GM1 to the incisures of Schmidt-Lanterman and the paranodal end segments of the myelin sheaths might indicate a role of gangliosides as adhesion molecules.     

The ultrastructure and functions of the ovariole sheath and tunica propria in the flour moth

The ultrastructure of the ovariole sheath, tunica propria, and associated cells is described. The sheath contains circular and longitudinal muscles and completely covers each ovariole except at the clefts between the oöcyte chambers where pores occur. Muscular movements cause haemolymph components to be pumped through these pores to the oöcyte at a rate faster than can be explained by diffusion. The tunica propria consists of two intercellular layers of fine granules and is not considered to have elastic properties. It may act simply as a valve for incoming haemolymph proteins. Directly beneath the sheath pores are found macrophages containing bacteria in various states of digestion. These may protect the oöcytes from bacterial infections. The interfollicular cells found between the chambers also contain bacteria and are considered to be mycetocytes.     

Messenger RNAs located in myelin sheath assembly sites

The targeting of mRNAs to specific subcellular locations is believed to facilitate the rapid and selective incorporation of their protein products into complexes that may include membrane organelles. In oligodendrocytes, mRNAs that encode myelin basic protein (MBP) and select myelin-associated oligodendrocytic basic proteins (MOBPs) locate in myelin sheath assembly sites (MSAS). To identify additional mRNAs located in MSAS, we used a combination of subcellular fractionation and suppression subtractive hybridization. More than 50% of the 1,080 cDNAs that were analyzed were derived from MBP or MOBP mRNAs, confirming that the method selected mRNAs enriched in MSAS. Of 90 other cDNAs identified, most represent one or more mRNAs enriched in rat brain myelin. Five cDNAs, which encode known proteins, were characterized for mRNA size(s), enrichment in myelin, and tissue and developmental expression patterns. Two of these, peptidylarginine deiminase and ferritin heavy chain, have recognized roles in myelination. The corresponding mRNAs were of different sizes than the previously identified mRNA, and they had tissue and development expression patterns that were indistinguishable from those of MBP mRNA. Three other cDNAs recognize mRNAs whose proteins (SH3p13, KIF1A, and dynein light intermediate chain) are involved in membrane biogenesis. Although enriched in myelin, the tissue and developmental distribution patterns of these mRNAs differed from those of MBP mRNA. Six other cDNAs, which did not share significant sequence homology to known mRNAs, were also examined. The corresponding mRNAs were highly enriched in myelin, and four had tissue and developmental distribution patterns indistinguishable from those of MBP mRNA. These studies demonstrate that MSAS contain a diverse population of mRNAs, whose locally synthesized proteins are placed to contribute to myelin sheath assembly and maintenance. Characterization of these mRNAs and proteins will help provide a comprehensive picture of myelin sheath assembly.