Internodal microvilli of Schwann cells of myelinated fibres in lizard spinal roots project onto unmyelinated axons

Summary Tufts of microvilli originating from the internodal cytoplasm of Schwann cells associated with myelinated axons in apparently normal lizard spinal roots have been studied under the electron microscope by means of both single and serial sections. More than one tuft of internodal microvilli may arise from a single Schwann cell. Sometimes mitochondria and more frequently an organelle resembling a multivesicular body with a clear matrix can be found in the Schwann cell cytoplasm underlying a tuft of internodal microvilli. The dimensions (length: 0.4–1.0 m; diameter: 40–70 nm) and structure of internodal microvilli of the Schwann cell are very similar to those of nodal microvilli of the same cell. Each tuft of internodal microvilli projects towards an adjacent unmyelinated axon which at this site is partly devoid of its own Schwann cell sheath. Thus a single Schwann cell may be related to a myelinated axon and an unmyelinated axon at the same time. Patches of a dense axolemmal undercoating (which could be portions of the cytoskeleton) are present in the unmyelinated axon in close spatial correlation with internodal microvilli. The factors which could induce the formation of internodal microvilli as well as the possible role (or roles) of these microvilli are briefly discussed.     

Myelinated and unmyelinated primary afferent axons form contacts with cholinergic interneurons in the spinal dorsal horn

Cholinergic interneurons in laminae III/IV of the dorsal horn contain co-localised gamma-aminobutyric acid (GABA) and frequently form axoaxonic synapses with terminals of primary afferents. They are therefore probably last-order interneurons involved in presynaptic inhibition. The purpose of the present investigation was to determine if these cells receive direct input from primary afferents. Relationships between primary afferents and interneurons were investigated in adult rats. Myelinated primary afferents were labelled with the B-subunit of cholera toxin (CTb). Unmyelinated afferents were labelled with IB4 lectin and an antibody to identify calcitonin-gene-related peptide (CGRP). Cholinergic neurons were labelled with an antibody raised against choline acetyltransferase and examined with a confocal microscope. Cells were reconstructed with NeuroLucida for Confocal and afferent contacts plotted. Interneurons (N=30) received an average of 20.2+/-11.9 (SD) contacts from CTb-labelled primary afferents, which were preferentially distributed on proximal and intermediate dendrites. Interneurons with dendrites which extended into lamina II (N=20) received an average of 27.4+/-19.0 IB4 contacts (on intermediate and distal dendrites) and 9.2+/-6.8 CGRP contacts. It is concluded that cholinergic interneurons receive contacts from both myelinated and unmyelinated primary afferents and different classes of afferent target particular dendritic domains. Cholinergic interneurons are likely to be components of an inhibitory feedback pathway that is monosynaptically activated by primary afferents.     

Cytochalasin D disrupts the restricted localization of N-CAM, but not of L1, at sites of Schwann cell-neurite and Schwann cell-Schwann cell contact in culture

The neural recognition molecules L1 and N-CAM have been shown to be preferentially localized at sites of Schwann cell-to-neurite and Schwann cell-to-Schwann cell contact in vitro. In the present study, we investigated the mechanisms underlying the restricted expression of these molecules at the Schwann cell surface, focusing on the possible role of actin filaments. Co-cultures consisting of Schwann cells from newborn mice and explants of dorsal root ganglia from chicken embryos were maintained in the absence or presence of cytochalasin D, an agent disrupting actin filaments. Immunoelectron microscopy with mouse-specific antibodies was carried out to quantify the restricted localization of L1 and N-CAM at the Schwann cell surface in contact with neurites. After 2 days of co-culturing in the absence of cytochalasin D, approximately 65% of the cell–cell contacts showed a restricted immunoreactivity for L1 and N-CAM. The accumulation of L1 at contact sites was unchanged in cytochalasin D-treated co-cultures, while the agent strongly reduced the restricted localization of N-CAM to 20% of all cell–cell contacts. The disruption of N-CAM accumulation appeared to be rapid and occurred within 5 h of cytochalasin D treatment. These results indicate that the restricted localization of N-CAM, but not of L1, is sensitive to cytochalasin D treatment, suggesting a dependence on the integrity of the actin network. Thus, different mechanisms may regulate the subcellular distribution of cell adhesion molecules in Schwann cells.     

Selective vulnerability of unmyelinated fiber Schwann cells in nerves exposed to local anesthetics

When peripheral nerves of experimental rats are exposed to local anesthetics, distinctive and reproducible pathologic changes occur involving the perineurial sheath and endoneurial contents. Application of intermediate strength concentrations of the local anesthetics, 2-chloroprocaine, lidocaine, etidocaine, and intermediate or high concentrations of procaine to the surface of rat sciatic nerves resulted in the following changes. By 48 hours, the perineurial sheath exposed to the drug was disrupted and became permeable to granulocytes which infiltrated the subjacent endoneurium in conjunction with edema formation in the endoneurial interstitium. Application of 10% procaine to exposed nerve resulted in extensive demyelination. The most striking pathologic change occurring with either intermediate or high doses was accumulation of lipid droplets in Schwann cells, a phenomenon that occurred often in myelin-producing Schwann cells but much less frequently in unmyelinated fiber Schwann Cells. Lipid accumulation appears to be one of several reactive changes that affect Schwann cells of myelinated fibers and is dose-dependent. On the other hand, while reactive changes were infrequently seen in unmyelinated fiber Schwann cells, these cells appeared more susceptible to injury as shown by electron microscopy. Injury to Schwann cells by local anesthetics is temporary because these cells can replicate quickly. Autoradiographic studies of thymidine incorporation 1 week after procaine administration to the sciatic nerve showed intense proliferation of Schwann cells, but no such activity in controls. These findings support the view that their neurotoxic properties may account in some part for the function of local anesthetics, that Schwann cells of small unmyelinated fibers are more vulnerable to these agents than those of myelinated fibers, and that destruction of their supporting cells is followed by vigorous mitotic activity in the endoneurium.     

Myelination of regenerated axons in goldfish optic nerve by Schwann cells

Summary This study uses immunohistochemistry and EM to examine the site of injury in goldfish optic nerve during axonal regeneration. Within seven days of nerve crush axons begin to regrow and a network of GFAP⁺ reactive astrocytes appears in the nerve on either side of the injury. However, the damaged area remains GFAP^–. By 42 days after nerve crush, the sheaths of new axons acquire myelin marker 6D2, and the crush area becomes populated by a mass of longitudinally-orientated S-100⁺ cells. Ultrastructurally, the predominant cells in the crush area bear a strong resemblance to peripheral nerve Schwann cells; they display a one-to-one association with myelinated axons, have a basal lamina and are surrounded by collagen fibres. It is proposed that these cells are Schwann cells which enter the optic nerve as a result of crush, where they become confined to the astrocyte-free crush area.     

Schwann cells in the regenerating fish optic nerve: evidence that CNS axons, not the glia, determine when myelin formation begins

Fish optic nerve fibres quickly regenerate after injury, but the onset of remyelination is delayed until they reach the brain. This recapitulates the timetable of CNS myelinogenesis during development in vertebrate animals generally, and we have used the regenerating fish optic nerve to obtain evidence that it is the axons, not the myelinating glial cells, that determine when myelin formation begins. In fish, the site of an optic nerve injury becomes remyelinated by ectopic Schwann cells of unknown origin. We allowed these cells to become established and then used them as reporters to indicate the time course of pro-myelin signalling during a further round of axonal outgrowth following a second upstream lesion. Unlike in the mammalian PNS, the ectopic Schwann cells failed to respond to axotomy and to the initial outgrowth of new optic axons. They only began to divide after the axons had reached the brain. Shortly afterwards, small numbers of Schwann cells began to leave the dividing pool and form myelin sheaths. More followed gradually, so that by 3 months remyelination was almost completed and few dividing cells were left. Moreover, remyelination occurred synchronously throughout the optic nerve, with the same time course in the pre-existing Schwann cells, the new ones that colonised the second injury, and the CNS oligodendrocytes elsewhere. The optic axons are the only common structures that could synchronise myelin formation in these disparate glial populations. The responses of the ectopic Schwann cells suggest that they are controlled by the regenerating optic axons in two consecutive steps. First, they begin to proliferate when the growing axons reach the brain. Second, they leave the cell cycle to differentiate individually at widely different times during the ensuing 2 months, during the critical period when the initial rough pattern of axon terminals in the optic tectum becomes refined into an accurate map. We suggest that each axon signals individually for myelin ensheathment once it completes this process.     

Gamma interferon, but not Mycobacterium leprae, induces major histocompatibility class II antigens on cultured rat Schwann cells

Summary In order to investigate the possible role of Schwann cells in immune reactions, and in particular their involvement in the response to infection withMycobacterium leprae, it was determined under what conditions Schwann cells express major histocompatibility complex class II (MHC class II) antigens, since these molecules are thought to have a key role in antigen presentation during cellular immune responses. In situ andin vitro preparations from newborn and adult rat sciatic nerves were used as a model system to examine this question. Schwann cells in dissociated cell cultures did not express immunohistochemically detectable amounts of MHC class II antigens. Teased nerve preparations from the sciatic nerves of healthy adult rats showed no detectable immunolabelling of either myelin-forming or non-myelin-forming Schwann cells.When dissociated Schwann cell cultures derived from the sciatic nerves of either neonatal or adult rats were treated with 10, 50 or 100 units of gamma Interferon, MHC class II antigens were detectable on the surface of some Schwann cells 48 h after addition of the interferon. By 72 h, 32.29 ± 3.9% of Schwann cells in the cultures from neonatal rats and 53.32 ± 5.4% of Schwann cells in cultures from adult rats, identified by the presence of intracellular S-100, were clearly MHC class II-positive, especially at doses of 50 and 100 units per ml of gamma interferon. Some, but not all, of the fibroblastic cells were very weakly MHC class Il-positive. Infection of the cultures withMycobacterium leprae did not induce MHC class II antigen expression in either Schwann cells or fibroblasts.These results suggest that one of the functional roles of Schwann cells' is the presentation of foreign antigens to T lymphocytes during nerve infection, leading to activation or augmentation of the cellular immune response. With respect toMycobacterium leprae in particular, it is therefore possible that infected Schwann cells might be capable of participating in the normal immune response toMycobacterium leprae.     

Studies on unmyelinated axons and varicosities in the olfactory cortex

Summary The main afferent input to the olfactory cortex from the olfactory bulbs is via the lateral olfactory tract (LOT). The axons within the lateral olfactory tract are myelinated. On leaving the LOT, they lose their myelination as they fan out over the layer immediately beneath the pial surface to make en passant synaptic connections with dendrites from neurones within the olfactory cortex. Using the guinea-pig, a semiquantitative electron micrographical study was made of the density and dimensions of these unmyelinated axons and the varicosities they create. The unmyelinated axons were very fine (0.17 ± 0.004 m in diameter) and punctuated at 2 m intervals by varicosities containing a single type of vesicle. The electrophysiological consequences of this close varicosity spacing is that axonal and varicosity membranes behave electrically as single units.     

The structure of Schwann cells in unmyelinated fibres. A qualitative and quantitative electron microscope study

The structure, size and distribution of many cytoplasmic components of Schwann cells associated with unmyelinated axons in lizard thoracic spinal roots were analysed under the electron microscope. The percentages of Schwann cell cytoplasmic area occupied by the following cytoplasmic components were determined: mitochondria, Golgi apparatus, granular endoplasmic reticulum, multivesicular bodies, smooth endoplasmic reticulum, lipofuscin granules, peroxisome-like bodies, autophagic vacuoles, dense bodies and lipid droplets. A linear correlation was found between the sectional areas of the mitochondria and granular endoplasmic reticulum of the Schwann cell and both length of Schwann cell plasma membrane profile and size of the related axoplasm. The structure of Schwann cells associated with unmyelinated axons and that of Schwann cells associated with myelinated axons were compared in the same species and in the same region of the peripheral nervous system using the same fixative and the same preparation technique. Some differences were detected in the organization of the granular endoplasmic reticulum, in the presence of cilia and in the percentages of cytoplasm occupied by various components. The hypothesis that Schwann cell mitochondria and granular endoplasmic reticulum are involved in the production and storage of proteins for the plasma membrane of this cell as well as the hypothesis that these organelles are involved in the production and storage of protein metabolites which are subsequently transferred to the related axons seem applicable not only to Schwann cells associated with myelinated axons (Pannese et al., in press), but also to those associated with unmyelinated ones.     

Vesicular release of glutamate from unmyelinated axons in white matter

Directed fusion of transmitter-laden vesicles enables rapid intercellular signaling in the central nervous system and occurs at synapses within gray matter. Here we show that action potentials also induce the release of glutamate from axons in the corpus callosum, a white matter region responsible for interhemispheric communication. Callosal axons release glutamate by vesicular fusion, which induces quantal AMPA receptor-mediated currents in NG2(+) glial progenitors at anatomically distinct axo-glial synaptic junctions. Glutamate release from axons was facilitated by repetitive stimulation and could be inhibited through activation of metabotropic autoreceptors. Although NG2(+) cells form associations with nodes of Ranvier in white matter, measurements of conduction velocity indicated that unmyelinated fibers are responsible for glutamatergic signaling with NG2(+) glia. This activity-dependent secretion of glutamate was prevalent in the developing and mature mouse corpus callosum, indicating that axons within white matter both conduct action potentials and engage in rapid neuron-glia communication.     

Identification of unmyelinated axons in fractions from caudate-putamen in the rat

Summary Previous studies have indicated that fractions of unmyelinated axons can be obtained from cerebellum, whole brain or forebrain. It seemed likely that unmyelinated axons break into segments upon homogenization and that any circumscribed region of brain containing abundant unmyelinated axons could be found to yield axonal fractions. Caudate-putamen material was used to test this hypothesis. Fractions analogous to cerebellum were obtained from sucrose gradients and were found to yield abundant axonal profiles which could be correlated with the appearance of unmyelinated axons after sucrose immersion in situ. We conclude that fractions of unmyelinated axons can likely be obtained from any anatomical region rich in them and can be used upon purification for obtaining chemical data from different axonal types.Supported in part by NIH Grant 2-RO-1 NS-09745.     

Alveolar macrophages down-regulate local pulmonary immune responses against intratracheally administered T-cell-dependent, but not T-cell-independent antigens.

The role of alveolar macrophages in the pulmonary immune response against various antigens was studied after elimination of alveolar macrophages by intratracheal administration of liposome-encapsulated dichloromethylene diphosphanate. When the responses against T-cell-independent type 1 and type 2 antigens were compared, it was found that elimination of alveolar macrophages had no effect on T-cell-independent antigens. Intratracheal antigen administration resulted in low lung associated, local responses, although some response was observed in the spleen. In contrast, elimination of alveolar macrophages resulted in an increase in local pulmonary immune response against T-cell-dependent antigens. We conclude from these experiments that alveolar macrophages play an important role in controlling the local pulmonary immune response against T-cell-dependent antigens by down-regulation of local T-cell populations. The alveolar macrophages do not down-regulate the response against intratracheally administered T-cell-independent antigens, although they are important in the protection against inflammatory damage caused by bacterial endotoxins.     

Fructose supports energy metabolism of some, but not all, axons in adult mouse optic nerve

We used transmission electron microscopy (TEM) and electrophysiological techniques to characterize the morphology and stimulus-evoked compound action potential (CAP), respectively, of the adult mouse optic nerve (MON). Electrophysiological recordings demonstrated an identical CAP profile for each MON. An initial peak, smallest in area and presumably composed of the fastest-conducting axons displayed the lowest threshold for activation as expected for large axons. The second peak, the largest, was presumably composed of axons of intermediate diameter and conduction velocity, and the third peak was composed of the slowest and presumably smallest axons. In 10 mM fructose, the first CAP peak area was reduced by 78%, but the second and third peaks were unaffected. Histological analysis revealed a cross-sectional area of 33,346 microm2, containing 24,068 axons per MON. All axons were myelinated and axon diameter ranged from 0.09 to 2.58 microm, although 80 +/- 6% of the axons were <0.75 microm in diameter and only 0.6 +/- 0.3% of the axons were >2 microm in diameter. After bathing in fructose for 2 h 94 +/- 2% of normal appearing axons were <0.75 microm in diameter and none were >1.5 microm-all of the larger axons being grossly abnormal in structure. We conclude that fructose is unable to support function of the larger axons contributing to the first CAP peak, thus enabling us to identify a distinct population of axons that contributes to that peak.     

Presynaptic Na/Ca action potentials in unmyelinated axons of olfactory cortex

(1) Pial surface slices of guinea-pig olfactory cortex were cut to have a thickness of 150 m. Action potentials were recorded from the sectioned ends of the unmyelinated afferent axons originating from the lateral olfactory tract (LOT). These potentials were prolonged by the K-channel blocker 3,4-diaminopyridine (0.1 mmol/l) and further lengthened by tetraethylammonium (10 mmol/l). The action potential was also greatly prolonged by partly replacing the K⁺ in the bathing solution by Cs⁺. (2) These prolonged action potentials were shortened by Cd²⁺; Gd³⁺ (gadolinium); Ni²⁺; Mn²⁺; Co²⁺, in order of potency. The residual early component of the action potential was tetrodotoxi (TTX) sensitive. In contrast, the LOT action potential wal little affected by Ca-channel blockade. (3) Organic Ca-channel blockers either had no effect (0.05 mmol/l nifedipine), or depressed the early and later phases of the prolonged action potential equally (0.05–0.5 mmol/l verapamil or 0.05–0.2 mmol/l diltiazem). (4) a propagated action potential was also obtained in solution containing TTX and low Na⁺. This potential was supported by Ca²⁺, Sr²⁺ or Ba²⁺ and completely suppressed by Cd²⁺. (5) The later parts of the action potential, after K-channel blockade, had a pharmacological sensitivity towards Ca-channel blockers matching that of synaptic transmission. This suggests the falling phase of the action potential is caused by charge carrier (mainly Ca²⁺) passing through Ca-channels that have similar properties to, or are the same as those which open prior to transmitter release.     

Potential of Schwann cells from unmyelinated nerves to produce myelin: a quantitative ultrastructural and radiographic study

Summary In adult mice, most nerve fibres in the cervical sympathetic trunk (CST) are unmyelinated whereas a large proportion of sural nerve fibres are myelinated. This study of nerve grafts in syngeneic mice was designed to determine if Schwann cells originating from the unmyelinated CST would produce myelin when in contact with regenerating axons of the sural nerve. Quantitative microscopy of tritiated thymidine-labelled CST segments grafted to unlabelled sural nerve stumps revealed that, one month after grafting, previously unmyelinated grafts contained many myelinated fibres. By phase and electron microscope radioautography, nearly 40% of the myelin-producing cells in the reinnervated graft were shown to have originated in the unmyelinated CST. These findings indicate that Schwann cells originating from unmyelinated fibres are able to differentiate into myelin producing cells.     

Endopeptidase-24.11 is suppressed in myelin-forming but not in non-myelin-forming Schwann cells during development of the rat sciatic nerve.

Endopeptidase-24.11, which is identical with the common acute lymphoblastic leukemia antigen (CALLA), is a cell surface zinc metalloprotease that has the ability to hydrolyse a variety of physiologically active peptides. Interest in this enzyme is based on the view that it may play a role in the regulation of peptide signals in different tissues, including the nervous and immune systems. We have previously shown that endopeptidase-24.11 is present in Schwann cells in the peripheral nervous system of newborn pigs [Kioussi C. and Matsas R. (1991) J. Neurochem. 57, 431-440]. In the present study we have investigated the developmental expression of the endopeptidase by Schwann cells in the rat sciatic nerve, from embryonic day 16 to maturity. Endopeptidase-24.11 was monitored enzymatically as well as by immunoblotting and immunocytochemistry using the monoclonal anti-endopeptidase antibody 23B11. We found an age-dependent decline in both the enzyme activity and the levels of immunoreactive protein. Endopeptidase-24.11 was first detected at embryonic day 18 and was present in all neonatal and early postnatal Schwann cells. However, as myelination proceeded the endopeptidase was gradually suppressed in the majority of cells that form myelin but retained in non-myelin-forming cells in the adult animal. At this stage, only very few large diameter myelinated fibers expressed weakly endopeptidase-24.11. Schwann cells dissociated from postnatal day 5 nerves and cultured up to one week in the absence of axons expressed endopeptidase-24.11. These results show that the endopeptidase has a distinct developmental profile in the rat sciatic nerve, similar to that of a group of other Schwann cell surface antigens, including the cell adhesion molecules N-CAM and L1 and the nerve growth factor receptor. We suggest that, as is the case with these antigens, endopeptidase-24.11 may play a role in nerve development and/or regeneration. In addition, persistence of endopeptidase-24.11 in a minority of adult myelin-forming Schwann cells suggests a possible role for the enzyme in axon-myelin apposition and maintenance, especially of larger diameter axons.     

Loss of NF1 allele in Schwann cells but not in fibroblasts derived from an NF1-associated neurofibroma.

Neurofibromas, the hallmark of neurofibromatosis 1, are composed mainly of Schwann cells and fibroblasts. Inactivation of both NF1 alleles is the cause of these benign tumors, but it is unknown which cell type is the progenitor. In this study, we selectively cultured Schwann cells from an NF1-associated neurofibroma. Fibroblasts were also obtained by culturing the tumor cells under standard conditions. Using four intragenic markers, we genotyped the NF1 locus in the original tumor and in the derived Schwann cells and fibroblasts. Loss of heterozygosity for two informative markers, which indicates loss of one NF1 allele, was found in Schwann cells but not in fibroblasts. This result suggests that genetic alterations of the NF1 gene in Schwann cells are responsible for the development of neurofibromas.     

Schwann cell “necrosis” in unmyelinated nerve fibres of patients with polyneuropathy

Summary Five of eight patients with polyneuropathy showed coagulation necrosis in Schwann cells of unmyelinated fibres.

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Koagulationsnekrose der Schwannschen Zellen unmyelinisierter Nerven bei Patienten mit Polyneuropathien

Zusammenfassung 5 von 8 Patienten mit Polyneuropathie zeigten Koagulationsnekrosen der Schwannschen Zellen unmyelinisierten Nervenfasern.

     

The spatiotemporal relationship among Schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats

To morphologically define the aging-related features during muscle reinnervation the spatiotemporal relationships among the major components of the neuromuscular junctions (NMJs) were investigated. A total of 64 rats, 30 adults (4 months old) and 34 aged adults (24 months old), were used. Between 1 and 12 weeks after sciatic nerve-crushing injury, cryosections of skeletal muscle were single or double labeled for S100, a marker of Schwann cells (SCs), for protein gene product 9.5, a neuronal marker, and for alpha-bungarotoxin (alpha-BT), a marker of the acetylcholine receptor site (AChR site), and then observed by confocal laser microscopy. The most obvious age changes were noted: (1) the regenerating SCs and axons were delayed in their arrival at the NMJ, (2) the dimensions of terminal SCs and AChR sites displayed a drastic and long-lasting drop (for terminal SCs, during 1-8 weeks; for AChR sites, during 1-12 weeks); (3) the degree of spatial overlap between AChR sites and terminal SCs was markedly low until 8 weeks post-crush; (4) damage and poor formation in the SCs, terminal axons and AChR sites, together with poor process extension from the terminal SC or terminal axon, were pronounced; (5) persistent aberrant changes, such as multiple innervation and terminal axon sprouting, together with poorly formed collateral innervation, nerve bundles, and NMJs, more frequently occurred in the later reinnervation period. Thus, with aging, regeneration is impaired during the period in which regenerating SC strands and axons extend into NMJs and the subsequent establishment of nerve-muscle contact is in progress. A complex set of morphological abnormalities between or among the TSCs, terminal axons, and AChR sites may be important in slowing of regeneration and reinnervation in aged motor endplates.