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.     

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.     

Autoradiographic studies of uridine incorporation in peripheral nerve of the newt, Triturus

Autoradiographic studies combined with digestion tests of incorporated ³H-uridine showed that the peripheral nerve of Triturus contains ribonucleic acid. Localization studies revealed the presence of RNA in the axon, in the myelin and Schwann sheath, and in the Schwann cell body. Similar experiments on nerve separated by transection from its neuronal cell bodies yielded the same results. They showed that RNA of the nerve can be synthesized without the intervention of the neuronal cell body. The results strongly suggest that the radioactive substance, precursor or RNA, is transported inward from the Schwann cell to be deposited in the myelin sheath and axon. The route of passage and the possible sites of origin of the RNA in the nerve are discussed. A significant role is suggested for the Schmidt-Lantermann cleft because of its relations with the adaxonal layer of Schwann cytoplasm and with the myelin leaflets.     

THE GEOMETRY OF PERIPHERAL MYELIN SHEATHS DURING THEIR FORMATION AND GROWTH IN RAT SCIATIC NERVES

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

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.     

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.     

The ultrastructure of sensory corpuscles in the skin in the hedgehog snout

The ultrastructure of sensory nerve endings was examined in the snout skin in 3 adult hedgehogs (Erinaceus europaeus). The material was taken intravitally under total anaesthesia and processed in a usual way for the electron microscopy. The corpuscles were evaluated in the individual sections and series sections made through the whole corpuscle. In the superficial layers of the dermis simple sensory corpuscles and free endings were found. The simple sensory corpuscles can be divided into three types. a) Corpuscles containing a greater number of lamellae in the inner core, the lamellae are arranged regularly and are separated by two opposite clefts. The capsule is formed by only several lamellae undoubtedly of fibrocytic origin. b) Corpuscles containing a smaller number of wider lamellae in the inner core situated often at random. The clefts are also irregular and are often closed in the superficial layers of the inner core. The capsule is quite simple mostly formed by a single lamella of fibrocyte which often fails to form a continuous coat of the corpuscle. c) The third type is typical of its inner core being formed by few lamellae arranged irregularly. These corpuscles have no connective tissue capsule and are separated from the environments only by the basement membrane of superficial lamellae of the inner core. The corpuscles of the second type resemble considerably the developmental stages of simple sensory corpuscles as described in the literature in the cat. They are the same in size or smaller than the corpuscles of the first type. The free nerve endings occurred in two forms. a) Flattened (lanciform) nerve terminals. The axon is rich in mitochondria. The sides of the flattened terminal is lined with one to three wide lamellae while the axon reaches as far as the surface of the formation which is covered only with the basement membrane. b) Typical free endings rich in mitochondria which are embedded in the cytoplasm of Schwann cells or occasionally are covered only with the basement membrane. The lanciform endings which are not linked up with the hairs here may represent a transition from free endings to simple sensory corpuscles.     

THE ULTRASTRUCTURE OF ADULT VERTEBRATE PERIPHERAL MYELINATED NERVE FIBERS IN RELATION TO MYELINOGENESIS

Adult chameleon myelinated peripheral nerve fibers have been studied with the electron microscope in thin sections. The outer lamella of the myelin sheath has been found to be connected as a double membrane to the surface of the Schwann cell. The inner lamella is connected as a similar double membrane with the double axon-Schwann membrane. The relations of these double connecting membranes suggest that the layered myelin structure is composed of a double membrane which is closely wound about the axon as a helix. These findings support the new theory of myelinogenesis proposed recently by Geren. The possible significance of these results with respect to cell surface membranes and cytoplasmic double membranes is discussed.     

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)     

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.     

The fine structure of the rod-bipolar cell synapse in the retina of the albino rat

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mmicro in thickness and 400 mmicro in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mmicro in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.     

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 movements in mature mammalian peripheral nerve fibers

Mature mouse and cat peripheral nerve fibers have been examined in vitro by time-lapse photography. Some Schmidt-Lanterman clefts which were open at the start closed later; other were seen to open and then to close, some of them more than once. The implications of these movements are considered, especially in regard to the question of the passage of materials from the endoneurial connective tissue spaces to the axon. Myelin movements other than those occurring at the Schmidt-Lanterman clefts consisted primarily of the development and frequent regression of indentations of the myelin sheath. A single evagination was seen to develop and then to recede. These myelin movements suggest that previously described invaginations and evaginations of the myelin sheath, including flaps of “redundant myelin”, are not static but rather that they are in a state of movement, forming and regressing at intervals. The possible functional significance of the development and regression of myelin sheath indentations in relationship to axoplasmic flow is discussed.     

Diffusion Models for the Squid Axon Schwann Cell Layer

The Schwann cell, basement membrane, and connective tissue layers that surround the squid giant axon and constitute barriers to diffusion, were modeled in a number of ways to analyze various experimental results. The experiments considered are (a) the time-course of the potassium concentration in the space between the Schwann cell and the axon membrane (from now on referred to as the F-H space) after an initial loading, (b) the time-course of sodium concentration in the F-H space after a sudden change in the sodium concentration in the external fluid; (c) the time-course of the concentration of tetrodotoxin (TTX) or saxitoxin (STX) in the F-H space after a sudden change in external concentration, including (or not) the effects of specific binding of TTX or STX to sites on the axon membrane and nonsaturable binding to sites in the F-H space or in the spaces (clefts) between Schwann cells; (d) the effects of the F-H space, clefts, and diffusion into the clefts from the outside (from now on referred to as convergence into the clefts) on the measured series resistance.

The analysis shows that (1) in no case is it necessary to include the effects of the convergence into the clefts from the outside; (2) in case a, the basement membrane, connective tissue layers, and the unstirred layer may be neglected, i.e., the clefts are rate limiting; (3) in case b the clefts may be neglected, i.e., the unstirred layer is rate limiting; (4) in most cases the clefts may be replaced by an equivalent thin diffusion barrier.

     

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.     

The Fine Structure of the Rod-Bipolar Cell Synapse in the Retina of the Albino Rat

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mµ in thickness and 400 mµ in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mµ in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.     

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.     

Microfibrillar structure of growing cell wall in a coenocytic green alga,Boergesenia forbesii

A fine structure of cell wall lamellae in a coenocytic green algaBoergesenia forbesii was examined by electron microscopy. The wall has a polylamellate structure containing cellulose microfibrils 25 to 30 nm in diameter. The outer surface of the cell was covered by a thin structureless lamella, underneath which existed a lamella containing randomly-oriented microfibrils. The major part of the wall consisted of two types of lamellae, multifibrillar lamella and a transitional, matrix-rich one. In the former, microfibrils were densely arranged more or less parallel with each other. In the transitional lamella, existing between the multifibrillar ones, the microfibril orientation shifted about 30° within the layer. The fibril orientation also shifted 30° between adjacent transitional and multifibrillar layers, and consequently the microfibril orientation in the neighboring multifibrillar layers shifted 90°. It was concluded that the orientation rotated counterclockwise when observed from inside the cell. Each lamella in the thallus wall become thinner with cell expansion, but no reorientation of microfibrils in the outer old layers was observed. In the rhizoid, the outer lamellae sloughed off with the tip growth.     

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.     

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.