Distribution of the myelin-associated glycoprotein and P0 protein during myelin compaction in quaking mouse peripheral nerve

Ultrastructural studies have shown that during early stages of Schwann cell myelination mesaxon membranes are converted to compact myelin lamellae. The distinct changes that occur in the spacing of these Schwann cell membranes are likely to be mediated by the redistribution of (a) the myelin-associated glycoprotein, a major structural protein of mesaxon membranes; and (b) P0 protein, the major structural protein of compact myelin. To test this hypothesis, the immunocytochemical distribution of these two proteins was determined in serial 1-micron-thick Epon sections of ventral roots from quaking mice and compared to the ultrastructure of identical areas in an adjacent thin section. Ventral roots of this hypomyelinating mouse mutant were studied because many fibers have a deficit in converting mesaxon membranes to compact myelin. The results indicated that conversion of mesaxon membranes to compact myelin involves the insertion of P0 protein into and the removal of the myelin-associated glycoprotein from mesaxon membranes. The failure of some quaking mouse Schwann cells to form compact myelin appears to result from an inability to remove the myelin-associated glycoprotein from their mesaxon membranes.     

Schwann cell myelination requires timely and precise targeting of P(0) protein

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.     

Some Neuron-Hypodermis Relationships in the Parasitic Nematode Trichurus myocastoris Enigk

The association of neurons and hypodermal cells in longitudinal nerve chords and in interchordal positions (perhaps dorso-ventral commissures) in the body wall of Trichuris myocastoris Enigk has been examined with the electron microscope. Nerve chords are composed of several groups of neurons separated by processes of hypodermal cells; such groups may be functionally separate. Nerve cell bodies occur within the chords, and appear to be at least bipolar. Neurons emerge from the median chords and enter the interchordal regions, showing progressive invagination in hypodermal tissue with increased distance from the median line. Such invagination results in formation of mesaxons and may be significant in the nutrition of the neuron. Mesaxons may branch and may enclose single or groups of neurons; no spiralling of the mesaxon was seen. Occasionally more than one mesaxon was traced to a single neuron.     

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.     

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.     

The Structure of Myelin Sheaths in the Central Nervous System of Xenopus laevis (Daudin)

The structure of myelinated nerve fibres has been studied in the spinal cord and optic nerve of the tadpoles of Xenopus laevis. Potassium permanganate-fixed material was examined with the electron microscope. The myelin sheath itself is made up of spirally arranged lamellae in which the intraperiod and dense lines alternate. Inside the myelin sheath an inner cytoplasmic process surrounds the axon and where the external surfaces of its bounding membrane come together an internal mesaxon is formed. The intraperiod line begins within the mesaxon and the dense line usually begins in the same region by apposition of the cytoplasmic surfaces of the membrane. The width of each lamella is 140 A. The outer line in the sheath is the dense line and this terminates in a tongue where the cytoplasmic surfaces of the myelin-forming glial cell separate. Thus, central myelin in Xenopus tadpoles is arranged in the same way as peripheral myelin, the only difference being that in central fibres, cytoplasm on the outside of the sheath is confined to that present in the tongue. For this reason adjacent central sheaths come into apposition without any intervening material being present. When this occurs an intraperiod line is formed between them.     

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.     

Psychosine-induced changes in the neuromuscular transmission and structure of the neuromuscular junction in the frog

A decrease in the amplitude of the miniature and evoked end-plate potentials, as well as a change in the course of facilitation and depression of the end-plate potentials under rhythmic stimulation, were observed in psychosine-treated preparations of the cutaneous-pectoral muscle of the frog. The results of electron microscopic investigations indicate changes in the structure of synaptic Schwann cells enveloping the motor terminals and disturbances of the inner mesaxon structure of the myelinated axons.A. A. Ukhtomskii Institute of Physiology, Saint Petersburg University. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 482–490, July–August, 1992.     

Electron Microscopy of Peripheral Nerves and Neuromuscular Junctions in the Wasp Leg

The peripheral nerve branch innervating the femoral muscles of the common yellow jacket (Vespula carolina) has been found to possess a thick lemnoblast basement membrane and a complex mesaxon. The term "tunicated nerve" is proposed to designate the type of peripheral nerve in which one or several axons are loosely mantled by meandering, cytoplasm-enclosing membranes of the lemnoblast. The peripheral axon courses longitudinally in a groove in the muscle fiber between the plasma membrane of the muscle fiber and a cap formed by lemnoblast and tracheoblast. The junction is characterized by apposition of plasma membranes of axon and muscle fiber, abundant mitochondria, and synaptic vesicles in the axon, and aggregates of "aposynaptic granules" plus mitochondria and endoplasmic reticulum on the muscle side of the synapse. Unlike the vertebrate striated muscle fiber, no complex infolding of the synapsing plasma membrane of the muscle fiber occurs. The "connecting tissue" of the insect is formed by tracheoblasts, their basement membranes, and the basement membranes of other cells. Further mechanical support is given by the ramifying tracheoles. The physiologic roles of the specialized structures are considered.     

Neuromuscular Junctions in Flight and Tymbal Muscles of the Cicada

The tymbal muscle fiber in the cicada closely resembles the indirect flight muscle fiber in its structural detail. We agree with other authors that the tymbal muscle is a modified indirect flight muscle. The peripheral nerve branches to the tymbal and flight muscle fibers are similar to those in the wasp leg. The axon is loosely mantled by irregular turns of the mesaxon, enclosing cytoplasm. The nerve is therefore a tunicated nerve. The neuromuscular junction in the high frequency muscle fibers shows direct apposition of plasma membranes of axon and muscle fiber, large numbers of mitochondria and synaptic vesicles in the axon, and concentrations of mitochondria, aposynaptic granules, and endoplasmic reticulum in the postsynaptic area of the muscle fiber. Of special interest is the multitude of intracellular, opposing membranes in the postsynaptic area. They form laminated stacks and whorls, vesicles, cysternae, and tubules. They occasionally show continuity with the plasma membrane, the outer nuclear envelope, and the circumfibrillar endoplasmic reticulum. The membrane system in this area is designated "rete synapticum." It is believed to add to the electrical capacity of the neuromuscular junction, to serve in transmission of potentials, and possibly is the site of the oscillating mechanism in high-frequency muscle fibers.     

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.     

Internodal specializations of myelinated axons in the central nervous system

We have examined the localization of contactin-associated protein (Caspr), the Shaker-type potassium channels, Kv1.1 and Kv1.2, their associated beta subunit, Kvbeta2, and Caspr2 in the myelinated fibers of the CNS. Caspr is localized to the paranodal axonal membrane, and Kv1.1, Kv1.2, Kvbeta2 and Caspr2 to the juxtaparanodal membrane. In addition to the paranodal staining, an internodal strand of Caspr staining apposes the inner mesaxon of the myelin sheath. Unlike myelinated axons in the peripheral nervous system, there was no internodal strand of Kv1.1, Kv1.2, Kvbeta2, or Caspr2. Thus, the organization of the nodal, paranodal, and juxtaparanodal axonal membrane is similar in the central and peripheral nervous systems, but the lack of Kv1.1/Kv1.2/Kvbeta2/Caspr2 internodal strands indicates that the oligodendrocyte myelin sheaths lack a trans molecular interaction with axons, an interaction that is present in Schwann cell myelin sheaths.     

ELECTRON MICROSCOPIC OBSERVATIONS OF THE OLFACTORY MUCOSA AND OLFACTORY NERVE

The olfactory receptor cell is characterized by a distal process (the dendrite) which terminates in the olfactory passage as the olfactory rod. The olfactory rod is provided with numerous cilia which are similar in structure to those seen in other tissues. The central processes of the bipolar cell constitute the fila olfactoria. The cytoplasmic organelles of the sustentacular cell are concentrated at the apical and basal ends of the cell with a paucity of cytoplasmic elements in the region of the nucleus. The plasma membrane of the supporting cell forms a mesaxon for both the dendrite and axon of the bipolar cell. Terminal bars are present in the epithelial cells. The axons constituting the fila olfactoria form fascicles which are ensheathed by mesaxons of adjacent Schwann cells. Thus the olfactory neurons are ensheathed throughout their course by the membranes of sustentacular and Schwann cells. Observations of the olfactory mucosa with the electron microscope are discussed with respect to recent electrophysiological studies.     

The ultrastructure of neuromuscular and neural-neural relationships in in vitro maintained Dirofilaria Immitis

Relationships of neuromuscular junctions of the somatic musculature and associated neural-neural synapses in the ventral nerve trunk of the canine adult heartworm, Dirofilaria immitis, were studied by transmission electron microscopy. The heartworms were maintained in vitro prior to study. Nerve fibres in the trunk were highly invaginated into the cytoplasm of hypodermal cells and connected through the intercellular spaces via mesaxons. The nerve fibres contained neurotubules, neurofilaments and ribosomes. The nerve trunk and the muscle arms were separated by an epineurium averaging 250 nm in width. At the junctional site, a marked reduction in width of the epineurium was noted at the synaptic cleft. Often when two adjacent nerve fibres had adjacent neuromuscular junctions, an axo-axonal synapse and common mesaxon between the adjacent fibres were present. Varicosities were evident on some cross-sections through nerve fibres and ranged from a simple outward swelling against the muscle arm mass to exaggerated outgrowths measuring several micrometers in length.     

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.     

The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves

Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G(-/-) mice, these proteins "piled up" at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface.     

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.     

Cellular and Subcellular Distribution of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Its mRNA in the Rat Central Nervous System

The 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the "incisure-like" membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS.     

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.     

Fine structure of synapses in the dorsal nucleus of the lateral geniculate body of normal and blinded rats

Summary Degenerating boutons, observed from 2 to 60 days after eye enucleation, displayed decreased plasma membrane density, increased axoplasmic density, and enlarged mitochondria with deformed cristae when compared with boutons from normal animals. There was also a loss of synaptic plasma membrane specialization and the boutons abnormally indented contiguous dendrites. The number and appearance of synaptic vesicles in some degenerating boutons were notably altered. Phagocytosis of boutons in most instances appeared to be accomplished by astrocytes. When degeneration was first apparent in some boutons, the subsynaptic organelle in the adjacent dendritic cytoplasm was enlarged, somewhat less dense and was associated with small granular and circular profiles. Subsynaptic organelles in experimental animals were absent from contiguities between dendrites and other cell processes, except in a few instances when only small portions of boutons remained at their synaptic sites, suggesting that the organelles disappeared when boutons had been completely phagocytized.Degenerating myelinated axons, observed from 2 to 300 days after enucleation, exhibited the same triad of features as degenerating boutons. They appeared to be phagocytized in most instances by dense glial processes, presumably oligodendrocytic, which were normally situated between the axon and its myelin sheath and were related to the inner mesaxon.This investigation was supported by U.S.P.H.S. Training Grants Nos. 2 T1 GM 202 T1 CA 505506, and 2RO 1 AM 368806.The author expresses his appreciation to Dr. A. J. Ladman for acquainting him with the techniques used in the study and to Dr. R. J. Barrnett for valuable criticism of this report. Gratitude is also extended to Mr. E. Z. Rutkowski for making the drawing.