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.     

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 FINE STRUCTURE OF NERVE CELL BODIES AND THEIR MYELIN SHEATHS IN THE EIGHTH NERVE GANGLION OF THE GOLDFISH

The eighth cranial nerve ganglion consists of bipolar nerve cell bodies each occupying part of an internodal segment. The perikaryal sheaths range from a single layer of Schwann cell cytoplasm on the smallest cells to typical thick compact myelin on the largest. On most perikarya, the sheath displays an intermediate form, consisting of multiple layers of Schwann cell cytoplasm (loose myelin), or of loose and compact myelin continuous with each other. Internodes beyond the one containing the cell body bear only compact myelin. In loose myelin the thickness of each layer of Schwann cell cytoplasm is about 100 A. It may be much greater (~ 3000 A) particularly in the outermost layers of the sheath, or the cytoplasm may thin and even disappear with formation of a major dense line. The cytoplasmic layers are separated from each other by a light zone, 40 to 200 A wide, which in its broader portions may contain an intermediate line. Desmosomes sometimes occur between lamellae. In addition to the usual organelles, the perikaryal cytoplasm contains granular and membranous inclusions. Large cells covered by compact myelin have a consistently higher concentration of neurofilaments, and some of the largest cells, in addition, show a reduced concentration of ribosomes. The functional significance and possible origins of perikaryal myelin sheaths are discussed.     

THE FINE STRUCTURE OF ACOUSTIC GANGLIA IN THE RAT

Nerve cell bodies in the spiral and vestibular ganglia of the adult rat are surrounded by thin (about ten lamellae) myelin sheaths which differ in several respects from typical axonal myelin. In some instances lamellae surrounding perikarya appear as typical major dense lines, and in others as thin Schwann cell sheets in which cytoplasm persists. Discontinuities and irregularities appear in the structure of perikaryal myelin. Lamellae may terminate anywhere within the sheaths; they may bifurcate; they may reverse their direction; or they may merge with each other. The number of lamellae varies from one part of a sheath to another. In addition, the myelin of a single perikaryal sheath may receive contributions from more than one Schwann cell, which overlap and interleave with each other. The ganglion cells are of two types: those which are densely packed with the usual cytoplasmic organelles but have few neurofilaments (granular neurons), and those which exhibit large areas containing few organelles but have a high concentration of neurofilaments (filamented neurons). The latter cell type is ensheathed by myelin which is generally more compact that that surrounding the former. The formation and the physiologic significance of perikaryal myelin are discussed.     

Myelinated axons of ganglion cells in the retinae of teleosts

Summary In the retina of the goldfish and the rainbow trout, the axons of ganglion cells belong to the unmyelinated or the myelinated types. The unmyelinated fibers are either arranged in bundles in direct contact with neighboring fibers or they are separated by intervening lamellae of oligodendroglial cytoplasm. The myelin sheaths of the myelinated fibers differ greatly in thickness. Most fibers show 3 to 5 myelin layers; single fiber elements, however, show 10 or even more layers.     

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.     

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.     

The need for speed. II. Myelin in calanoid copepods

Speed of nerve impulse conduction is greatly increased by myelin, a multi-layered membranous sheath surrounding axons. Myelinated axons are ubiquitous among the vertebrates, but relatively rare among invertebrates. Electron microscopy of calanoid copepods using rapid cryofixation techniques revealed the widespread presence of myelinated axons. Myelin sheaths of up to 60 layers were found around both sensory and motor axons of the first antenna and interneurons of the ventral nerve cord. Except at nodes, individual lamellae appeared to be continuous and circular, without seams, as opposed to the spiral structure of vertebrate and annelid myelin. The highly organized myelin was characterized by the complete exclusion of cytoplasm from the intracellular spaces of the cell generating it. In regions of compaction, extracytoplasmic space was also eliminated. Focal or fenestration nodes, rather than circumferential ones, were locally common. Myelin lamellae terminated in stepwise fashion at these nodes, appearing to fuse with the axolemma or adjacent myelin lamellae. As with vertebrate myelin, copepod sheaths are designed to minimize both resistive and capacitive current flow through the internodal membrane, greatly speeding nerve impulse conduction. Copepod myelin differs from that of any other group described, while sharing features of every group. Accepted: 8 January 2000     

Ultrastructural changes of human sural nerves in the neuropathy induced by intrauterine methylmercury poisoning (so-called fetal Minamata disease).

Biopsy of the sural nerve was performed on three patients with severe Minamata disease of more than 10 years duration. There were so many unmyelinated and poorly myelinated nerve fibers that myelinated fibers scattered irregularly in small numbers or in groups of peculiar features in the intraneural bundle. Abnormaly thin or poorly formed myelin sheaths were noticed. Incomplete myelination and abnormal myelination varied in size and shape appeared as fetal anomaly. Regenerated axons extremely small in size remained singly or in groups following regenerative sprouting. Sometimes, extremely small axons with normal myelination were noticeable, while the axons were lost, leaving myelin sheaths. Axons occasionally contained increased neurofilaments. Schwann cells were not so increased as in adult Minamata disease. Degenerative changes of nerve fibers still proceeded, presumably because the patients lived in the mercury-contaminated district. Myelin degenerations and glycogen deposits in the axoplasm were identified.     

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.     

Incorporation of glycoproteins into peripheral nerve myelin

Peripheral nerve myelin contains a dominant low molecular weight glycoprotein called Po. To study the metabolism of this glycoprotein, tritiated fucose was injected into the peripheral nerves of adult mice and developing rats, and the temporal distribution of label was examined by autoradiography and gel electrophoresis. Mice and rat pups, injected with fucose, were sacrificed from 1 h to 98 days later. Series of autoradiographs were prepared. At the shortest labeling periods, newly formed product was confined to juxtanuclear Schwann cell cytoplasm, in association with regions rich in Golgi apparatus. After longer labeling periods, silver grain levels in Schwann cell cytoplasm decreased; concomitantly, there was an increase of silver grains associated with myelin. In adult animals, label associated with myelin was concentrated over outer layers of thickly myelinated fibers. Even at the longest time intervals examined (72 and 98 days), this distribution of label was largely retained. In contrast, in developing animals, label became associated with inner layers of the thicker sheaths. At no time was label observed over axons. Gel electrophoresis revealed that tritiated fucose was a suitable precursor for the faster migrating peripheral nerve glycoprotein(s). At all times examined, there was a single major peak of radioactivity that co-migrated on sodium dodecyl sulfate (SDS) acrylamide gels with the Po protein. Sometimes, a faster migrating shoulder of radioactivity was noted. With increased labeling periods, there was an enrichment of radioactivity associated with Po, indicative of a relatively slow turnover rate.     

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.     

Alteration of ethanolamine glycerophospholipid turnover in trembler dysmyelinating mutant: An analysis of the sciatic nerve by biochemistry and radioautography

The turnover of phospholipids was compared in peripheral nerves of Trembler dysmelinating mutant and control mice, after intraperitoneal and local injection of labeled ethanolamine. In the mutant sciatic nerve, neurochemical analysis showed that [¹⁴C]ethanolamine is incorporated into EGP (ethanolamine glycerophospholipids) of the sciatic nerve at a much higher rate in Trembler mutant than in control mice. Furthermore the decay rate of ¹⁴C-labeled EGP is faster in Trembler than in normal animals. The accelerated turnover of EGP in Trembler sciatic nerve affects the diacyl-EGP while the renewal of the alkenylacyl-EGP (plasmalogens) is slower than in controls. Quantitative radioautographic study at the ultrastructural level corroborate that the initial increase of the label in Trembler nerve fibers was different in axons, Schwann cells and myelin sheaths. EM radioautographs reveal indeed that the high label content observed in Trembler axons takes place preferentially in the myelinated portions of axons and drops within 1 week. In both myelinated and unmyelinated segments of the axons, the majority of the radioactivity was contained in axolemma and smooth axoplasmic reticulum. The 10-fold increase of label found in the myelin sheath of Trembler nerve fibers at 1 day raises the question of the origin of the labeled EGP, either by a stimulated synthesis in Schwann cells or by transfer from axonally transported phospholipids. In contrast, the label of axons, Schwann cells and myelin sheaths of control nerve remains stable during the same period.     

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 µ 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.     

Optical measurement of conduction in single demyelinated axons

Demyelination was initiated in Xenopus sciatic nerves by an intraneural injection of lysolecithin over a 2-3-mm region. During the next week macrophages and Schwann cells removed all remaining damaged myelin by phagocytosis. Proliferating Schwann cells then began to remyelinate the axons, with the first few lamellae appearing 13 d after surgery. Action potentials were recorded optically through the use of a potential-sensitive dye. Signals could be detected both at normal nodes of Ranvier and within demyelinated segments. Before remyelination, conduction through the lesion occurred in only a small fraction of the fibers. However, in these particular cases we could demonstrate continuous (nonsaltatory) conduction at very low velocities over long (greater than one internode) lengths of demyelinated axons. We have previously found through loose patch clamp experiments that the internodal axolemma contains voltage-dependent Na+ channels at a density approximately 4% of that at the nodes. These channels alone, however, are insufficient for successful conduction past the transition point between myelinated and demyelinated regions. Small improvements in the passive cable properties of the axon, adequate for propagation at this site, can be realized through the close apposition of macrophages and Schwann cells. As the initial lamellae of myelin appear, the probability of success at the transition zone increases rapidly, though the conduction velocity through the demyelinated segment is not appreciably changed. A detailed computational model is used to test the relative roles of the internodal Na+ channels and the new extracellular layer. The results suggest a possible mechanism that may contribute to the spontaneous recovery of function often seen in demyelinating disease.     

Localization of Phospholipid Synthesis to Schwann Cells and Axons

Quantitative electron microscopic autoradiography was used to detect and characterize endoneurial sites of lipid synthesis in mouse sciatic nerve. Six tritiated phospholipid precursors (choline, serine, methionine, inositol, glycerol, and ethanolamine) and a protein precursor (proline) were individually injected into exposed nerves and after 2 h the mice were perfused with buffered aldehyde. The labeled segments of nerve were prepared for autoradiography with procedures that selectively remove nonincorporated precursors and other aqueous metabolites, while preserving nerve lipids (and proteins). At both the light and electron microscope levels, the major site of phospholipid and protein synthesis was the crescent-shaped perinuclear cytoplasm of myelinating Schwann cells. Other internodal Schwann cell cytoplasm, including that in surface channels, Schmidt-Lanterman incisures, and paranodal regions, was less well labeled than the perinuclear region. Newly formed proteins were selectively located in the Schwann cell nucleus. Lipid and protein formation was also detected in unmyelinated fiber bundles and in endoneurial and perineurial cells. Tritiated inositol was selectively incorporated into phospholipids in both myelinated axons and unmyelinated fibers. Like inositol, glycerol incorporation appeared particularly active in unmyelinated fibers. Quantitative autoradiographic analyses substantiated the following points: myelinating Schwann cells dominate phospholipid and protein synthesis, myelinated axons selectively incorporate tritiated inositol, phospholipid precursors label myelin sheaths and myelinated axons better than proline.     

FINE STRUCTURAL LOCALIZATION OF CHOLESTEROL-1,2-3H IN DEGENERATING AND REGENERATING MOUSE SCIATIC NERVE

The localization of ³H-labeled cholesterol in nerves undergoing degeneration and regeneration was studied by radioautography at the electron microscope level. Two types of experiments were carried out: (a) Cholesterol-1,2-³H was injected intraperitoneally into suckling mice. 5 wk later, Wallerian degeneration was induced in the middle branch of the sciatic nerve, carefully preserving the collateral branches. The animals were then sacrificed at various times after the operation. During degeneration, radioactivity was found over myelin debris and fat droplets. In early stages of regeneration, radioactivity was found in myelin debris and regenerating myelin sheaths. Afterwards, radioactivity was found predominantly over the regenerated myelin sheaths. Radioactivity was also associated with the myelin sheaths of the unaltered fibers, (b) Wallerian degeneration was induced in the middle branch of the sciatic nerves of an adult mouse, preserving the collateral branches. Cholesterol-1,2-³H was injected 24 and 48 hr after the operation and the animal was sacrificed 6 wk later. Radioactivity was found in the myelin sheaths of the regenerated and unaltered fibers. The results from these experiments indicate that: (a) exogenous cholesterol incorporated into peripheral nerve during myelination remains within the nerve when it undergoes degeneration. Such cholesterol is kept in the myelin debris as an exchangeable pool from which it is reutilized for the formation of the newly regenerating fibers, especially myelin. (b) exogenous cholesterol incorporated into the nerves at the time that degeneration is beginning is also used in the formation of new myelin sheaths during regeneration, (c) mature myelin maintains its ability to incorporate cholesterol.     

A TIME-SEQUENCE AUTORADIOGRAPHIC STUDY OF THE IN VIVO INCORPORATION OF [1, 2-3H]CHOLESTEROL INTO PERIPHERAL NERVE MYELIN

A time-sequence study of the incorporation and distribution of cholesterol in peripheral nerve myelin was carried out by electron microscope autoradiography. [1,2-³H]Cholesterol was injected into 10-day old mice and the sciatic nerves were dissected out at 10, 20, 40, 60, 90, 120, and 180 min after the injection. 20 min after injection the higher densities of grains due to the presence of [³H]cholesterol were confined to the outer and inner edges of the myelin sheath. Practically no cholesterol was detected in the midzone of the myelin sheath. 1 ½ h after injection, cholesterol showed a wider distribution within the myelin sheath, the higher densities of grains occurring over the two peripheral myelin bands, each approximately 3,100 Å wide. Cholesterol was also present in the center of the myelin sheath but to a considerably lesser extent. 3 h after injection cholesterol appeared homogeneously distributed within the myelin sheath. Schwann cell and axon compartments were also labeled at each time interval studied beginning 20 min postinjection. These observations indicate that preformed cholesterol enters myelin first and almost simultaneously through the inner and outer edges of the sheath; only after 90 min does the density of labeled cholesterol in the central zone of myelin reach the same density as that in the outer and inner zones. These findings suggest that cholesterol used by the nerve fibers in the formation and maintenance of the myelin sheath enters the lamellae from the Schwann cell cytoplasm and from the axon. The possibility of a bidirectional movement of molecules, i.e. from the Schwann cell to the axon and from the axon to the Schwann cell through the myelin sheath, is noted. The results are discussed in the light of recent observations on the exchange, reutilization, and transaxonal movement of cholesterol.     

N-WASP is required for membrane wrapping and myelination by Schwann cells

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.