Schwann cell myelin ensheathing C.N.S. axons in the nerve fibre layer of the cat retina

Summary In the retina of the cat the axons of the nerve fibre layer are unmyelinated and are provided with a C.N.S. myelin sheath only in the extraocular part of the optic nerve. The present study demonstrates that in the apparently normal cat retina close to the optic disc, some axons of the nerve fibre layer run for a short distance in the perivascular space of the retinal arteries. While coursing in the perivascular space, these C.N.S. axons become transiently myelinated by Schwann cells, which form a typical P.N.S. myelin sheath. These P.N.S. myelin sheaths terminate at a heminode in the transitional zone in which the C.N.S. axons penetrate the perivascular glial sheath in order to leave or to re-enter the nerve fibre layer. It is suggested that the Schwann cells, which elaborate the P.N.S. myelin around C.N.S. axons, are descendants of the Schwann cells of the perivascular autonomie nerves. The present study shows that Schwann cells are able to provide previously unmyelinated C.N.S. axons with a P.N.S. myelin sheath.     

Defective myelination in the optic nerve of the Browman-Wyse (BW) mutant rat

Summary The Browman-Wyse (BW) rat displays a spectrum of ocular abnormalities which include myelination by Schwann cells of retinal ganglion cell (RGC) axons within the retina. Immunohistochemical and ultrastructural studies of the optic nerves of adult BW rats (30–60 days of age) with myelinated intraretinal axons were performed. Although individual nerves displayed considerable morphological variability, all were characterized by an initial dysmyelinated proximal segment which was separated from a normally myelinated distal segment by a transitional junctional zone. The proximal segment contained axons which were predominantly unmyelinated: where myelination occurred, almost all sheaths were P_o-positive, proteolipid protein-negative, and the myelinating cell was a Schwann cell. In the distal segment the distribution of myelinated axons appeared to be normal, sheaths were PLP⁺, and the myelinating cell was an oligodendrocyte. Within the proximal segment, axons that were myelinated by Schwann cells were isolated by a basal lamina and expanded extracellular spaces from the bulk of other RGC axons within the optic nerve. Few carbonic anhydrase (CAII)⁺ or GalC⁺ oligodendrocytes were seen in proximal segments that contained Schwann cells: anti-CAII antibody stained atypical cells within the proximal segments which did not resemble CAII⁺ oligodendrocytes in the distal segment, and which were probably GalC^–. Astrocytes appeared normal throughout the length of the nerve, and there was no morphological specialization at the junctional zone similar to that at the lamina cribrosa. The possible source (s) of the intraneural Schwann cells, and the pathogenetic mechanisms underlying the aberrant myelination of RGC axons within the BW optic nerve are discussed.     

Characterization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fibre.

1. The effects of alpha-bungarotoxin, nicotine and muscarine on the Schwann cell membrane potential have been studied in the giant nerve fibre of the squid. The external application of alpha-bungarotoxin (10(-6), 10(-8), 10(-9) M) irreversibly blocks the long-lasting Schwann cell hyperpolarizations following the conduction of nerve impulse trains by the axon. It also blocks the Schwann cell hyperpolarizing response to the external application of carbamylcholine (10(-6)M) to the resting nerve fibre. 2. Externally applied D-tubocurarine (10(-5)M) protects against the irreversible action of alpha-bungarotoxin (10(-9)M) on the Schwann cell. Within 10 min of reimmersion in toxin-free sea water there is complete recovery of the Schwann cell hyperpolarizing response to carbamylcholine (10(-6)M) which had been initially abolished. 3. Nicotine (10(-6)M) induces a prolonged hyperpolarization of the Schwann cells in the resting nerve fibre, wheras at the same concentration, muscarine has no appreciable effect on the Schwann cell membrane potential. 4. None of these drugs, at the concnetrations utilized in the present study, had any appreciable effect on the resting and action potentials of the axon. 5. These findings show the presence of acetylcholine receptors of the nicotinic type in the Schwann cell membrane, and give further support to the hypothesis on the role of the acetylcholine system in the genesis of the long-lasting Schwann cell hyperpolarizations caused by the conduction of nerve impulse trains by the axon.     

TACE (ADAM17) inhibits Schwann cell myelination

Tumor necrosis factor-α-converting enzyme (TACE; also known as ADAM17) is a proteolytic sheddase that is responsible for the cleavage of several membrane-bound molecules. We report that TACE cleaves neuregulin-1 (NRG1) type III in the epidermal growth factor domain, probably inactivating it (as assessed by deficient activation of the phosphatidylinositol-3-OH kinase pathway), and thereby negatively regulating peripheral nervous system (PNS) myelination. Lentivirus-mediated knockdown of TACE in vitro in dorsal root ganglia neurons accelerates the onset of myelination and results in hypermyelination. In agreement, motor neurons of conditional knockout mice lacking TACE specifically in these cells are significantly hypermyelinated, and small-caliber fibers are aberrantly myelinated. Further, reduced TACE activity rescues hypomyelination in NRG1 type III haploinsufficient mice in vivo. We also show that the inhibitory effect of TACE is neuron-autonomous, as Schwann cells lacking TACE elaborate myelin of normal thickness. Thus, TACE is a modulator of NRG1 type III activity and is a negative regulator of myelination in the PNS.     

大鼠视神经切断后视网膜外网层突触可塑性研究

应用闪光视网膜电图(fERG)、细胞色素c氧化酶(CO)组织化学染色及突触囊泡素免疫荧光染色方法检测视神经切断术后,视网膜外网层相关神经元的功能活动及突触可塑性变化。结果显示:fERGb波峰潜伏期在视神经损伤后均显著延长;b波振幅在损伤第3、5d组增高,此后逐渐下降并在7、14、21d组低于正常。外网层CO活性在视神经切断第3、5d上调,此后逐渐下降,至第14、21d低于正常。外网层突触囊泡素阳性颗粒在视神经切断第5d增多,此后逐渐下降,至第14、21d低于正常。本研究结果提示视神经切断后,视网膜内外网层神经元的功能在早期将出现一过性的增强,结构也有相应的代偿性改变,随后即发生跨神经元的逆行性溃变。     

Evidence for an amacrine cell system in the ganglion cell layer of the rat retina

In the ganglion cell layer of the rat retina approx 50% of the cells with the Nissl morphology of neurons survive optic nerve section in infant and adult rats and cannot be retrogradely labelled with horseradish peroxidase. The number of neurons which can be retrogradely labelled with horseradish peroxidase from subcortical visual centres is similar to the number of axons in the optic nerve, and it is concluded that the small neurons do not send an axon into the optic nerve. The dendritic tree of the cells which have axons was demonstrated by filling the cells with horseradish peroxidase from the optic nerve. The dendritic structure of the cells which survive optic nerve section was shown by injecting horseradish peroxidase into the retina or impregnating with the Golgi method the cells which survive optic nerve section. A variety of amacrine cells were found in the ganglion cell layer which form branches in the lower part of the inner plexiform layer.It can be concluded that amacrine cells form a substantial number of the neurons in the ganglion cell layer.     

Effects of acetylcholine and carbamylcholine on the axon and Schwann cell electrical potentials in the squid nerve fibre

1. The effect of acetylcholine and carbamylcholine on the axon and Schwann cell membrane potential have been studied in the giant nerve fibre of the squid. The addition of carbamylcholine (10(-6)M) to the external sea-water medium has no appreciable effects on the resting and action potentials of the axon. However, it induces a long-lasting hyperpolarization in the surrounding Schwann cells of the unstimulated intact or slit nerve fibres which is completely blocked by D-tubocurarine (10(-9)M). Eserine (10(-9)M) prolongs the Schwann cell hyperpolarizations induced by a 1 min exposure of the unstimulated nerve fibres to acetylcholine (10(-7)M).2. The addition of carbamylcholine (10(-6)M) to the external medium increases the relative permeability of the Schwann cell membrane to the potassium ion in slit nerve fibres. Yet, a hundredfold reduction in external sodium concentration has no appreciable effect on the hyperpolarization of the Schwann cells of the slit nerve fibre under similar conditions.3. Tetrodotoxin at a concentration of 5 x 10(-8)M has no appreciable effects on either the Schwann cell electrical potential or on the hyperpolarizing action of carbamylcholine on the Schwann cells of the unstimulated intact nerve fibres.4. These findings indicate the presence of acetylcholine receptors in the plasma membrane of the Schwann cell in these nerve fibres and give further support to the hypothesis on the role of the cholinergic system in the genesis of the long-lasting Schwann cell hyperpolarizations caused by the conduction of nerve impulse trains by the axon.     

Membrane specialization and axo-glial association in the rat retinal nerve fibre layer: freeze-fracture observations

Summary The ultrastrucrure of non-myelinated ganglion cell axolemma within the retinal nerve fibre layer of adult rats was examined by thin section and freeze-fracture electron microscopy. Most of the axolemma within the nerve fibre layer does not exhibit any membrane specializations; intramembranous particles are partitioned with a density of 1750 m^–2 on the P-fracture face and 225 m^–2 on the E-face of the non-specialized axolemma. The nerve fibres also exhibit specialized foci of axolemma, at which the axons are abutted by the tips of blunt, radially oriented processes from Müller cells. At such sites of axo-glial association, an electron-dense undercoating is present beneath the axon membrane. Freeze-fracture analysis revealed a substantial increase in the density of E-face particles (>500 m^–2) at sites of association between the tips of blunt glial processes and the axon. These findings demonstrate that non-myelinated axolemma of the retinal nerve fibre layer can exhibit spatial heterogeneity, with patches of node-like membrane at regions of specialized association with glial cell processes. On the basis of their morphological similarity to nodes of Ranvier, we suggest that these specialized axon regions represent foci of inward ionic current.     

Events in degenerating cat peripheral nerve: Induction of Schwann cell S phase and its relation to nerve fibre degeneration

Summary Severance of a peripheral nerve leads to a characteristic series of events in the distal stump, including the dissolution of axons and myelin and the proliferation of Schwann cells within their basal lamina. This study examines the relationship between the spatial-temporal pattern of the induction of the Schwann cell S phase, loss of the structural and functional properties of axolemma, and the clearance of myelin debris in the cat tibial nerve. Nerve transection stimulated a monophasic increase in [³H]thymidine incorporation that peaked at 4 days post-transection throughout an 80-mm length of distal stump. Light microscope autoradiography revealed prominent incorporation into Schwann cells of myelinated fibres. Treatment of distal stumps with mitomycin C at the time of nerve transection greatly retarded thymidine incorporation and clearance of myelin debris, but not the time course of axonal degeneration, decline in the synthesis of the major myelin glycoprotein, P₀, or the onset of ovoid formation. Nerve transection also greatly reduced the specific uptake of [³H]saxitoxin (STX), a ligand which binds to voltage-sensitive sodium channels. Binding in the distal stump fell precipitously to 20% of the normal at 4 days post-transection, concurrent with the peak of thymidine incorporation. This low level of binding was maintained for periods of up to 70 days, demonstrating that some STX binds to structures other than axons in denervated distal stumps. Prior treatment with mitomycin C delayed the loss of specific STX binding. In conclusion, these studies suggest that: (a) Schwann cell DNA replication- and/or mitosis regulates other events during Wallerian degeneration, including myelin degeneration, catabolism of P₀ and the clearance of sodium channels from nodal axolemma; (b) the decline in P₀ synthesis and/or shift to synthesis of less extensively processed P₀ is independent of the induction of Schwann cell S phase; and (c) Schwann cells enveloping myelinated axons enter S phase within a 24-h period throughout the entire 80-mm length of distal stump.     

Allogeneic nerve grafts in the rat, with special reference to the role of Schwann cell basal laminae in nerve regeneration

Summary The role of basal laminae as conduits for regenerating axons in an allogeneic graft was examined by transplanting a 3 cm long segment of the sciatic nerve from the Brown Norway to the Fischer 344 strain of rat. These strains are not histocompatible with each other. In order to compare the nerve regeneration in variously treated grafts, three different types of graft were employed: non-treated (NT), predenervated (PD), and predenervated plus freeze-treated (PDC) grafts. The cytology of nerve regeneration through these grafts was examined by electron microscopy at four, seven, 14, 30 and 60 days after grafting.In the PDC graft, in which Schwann cells were dead on grafting, basal laminae were well preserved in the form of tubes after Schwann cells and myelin sheaths had been removed at seven days after grafting. Regenerating axons accompanied by immature host Schwann cells grew out through such basal lamina tubes in the same fashion as observed in our previous studies. By day 14, axons extended as far as the middle of the graft. In the proximal part they were separated into individual fibres and even thinly myelinated by Schwann cells.On the other hand, in the NT and PD grafts in which Schwann cells were alive on grafting, most Schwann cells and myelin sheaths appeared to undergo autolytic degeneration by day 14, while Schwann cell basal laminae were left almost intact in the form of tubes. A few regenerating axons were seen associated with Schwann cells in the proximal portion by day seven. It is probable that host Schwann cells moved into the graft after donor cells had been degraded. Schwann cell basal laminae tended to be damaged at the site of extensive lymphoid cell infiltration.By day 30, regenerating axons had arrived at the distal end of the graft in all three types of graft: in the PDC graft thick axons were fully myelinated, whereas in the PD graft they were only occasionally myelinated and in the NT graft most axons were still surrounded by common Schwann cells. By 60 days after grafting, regenerating axons were well myelinated in the host nerve as observed 1 cm distal to the apposition site in all the three types of graft.These findings show that Schwann cell basal laminae can serve as pathways (most efficiently in the PDC graft) for regenerating axons in a 3 cm long allograft in the rat.     

The mood stabilizer valproic acid induces proliferation and myelination of rat Schwann cells

Schwann cells (SCs) within peripheral nerve respond robustly after exposure to neurotrophic factors. Recent results have revealed that valproic acid (VPA), at a clinically relevant therapeutic concentration, produces effects similar to neurotrophic factors, and promotes neurite growth and cell survival. We hypothesized that VPA could also induce Schwann cell response. In this study, we sought to determine how pure Schwann cells responded to VPA by evaluating for proliferation, expression of S-100, growth cone-associated protein 43 (GAP-43), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP). Immunohistochemistry demonstrated that the Schwann cells were positive for S-100, GAP-43, MAG, and MBP greater than 99% of the experimental cells. The rate of proliferation was increased in experimental cells from MTT assay and Bromodeoxyuridine/DAPI double staining. Furthermore, Western blot showed an up-regulation in GAP-43, MAG and MBP protein expression in experimental cells, respectively. We also found that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) 1/2 pathway was involved in the enhanced cell proliferation of Schwann cells evoked by VPA. This study provides novel information regarding Schwann cell response to VPA, which might help the understanding of VPA-based treatment for peripheral nerve injury.     

Zonulae occludentes of the myelin lamellae in the nerve fibre layer of the retina and in the optic nerve of the rabbit: A demonstration by the freeze-fracture method

Summary Ridges and grooves composing extensive zonulae occludentes are revealed by the freeze-fracture method on split myelin lamellae in the nerve fibre layer of the retina and in the optic nerve of the rabbit. The junctions are located immediately internal to the outer loop of the myelin sheath and in corresponding areas of deeper myelin layers. They follow a straight or gently undulating course along the axis of the fibres. Only at the paranodal region of nodes of Ranvier do they deviate and assume a transverse course. The strands of these zonulae occludentes probably represent the radial thickenings of the intraperiod line described in thin sections.An abstract of this paper was presented at the Colloque Annuel de la Société Francaise de Microscopie Electronique, Rennes, 27–30 May 1974.     

Axon-schwann cell interaction in the squid nerve fibre

The electrical properties of Schwann cells and the effects of neuronal impulses on their membrane potential have been studied in the giant nerve fibre of the squid.1. The behaviour of the Schwann cell membrane to current injection into the cell was ohmic. No impulse-like responses were observed with displacements of 35 mV in the membrane potential. The resistance of the Schwann cell membrane was found to be approximately 10(3) Omega cm(2).2. A long-lasting hyperpolarization is observed in the Schwann cells following the conduction of impulse trains by the axon. Whereas the propagation of a single impulse had little effect, prolonged stimulation of the fibre at 250 impulses/sec was followed by a hyperpolarization of the Schwann cell that gradually declined over a period of several minutes.3. The prolonged effects of nerve impulse trains on the Schwann cell were similar to those produced by depolarizing current pulses applied to the axon by the voltage-clamp technique. Thus, a series of depolarizing pulses in the axon was followed by a long-lasting hyperpolarization of the Schwann cells. In contrast, the application of a series of hyperpolarizing 100 mV pulses at a frequency of 1/sec had no apparent effects.4. Changes in the external potassium concentration did not reproduce the long-lasting effects of nerve excitation.5. The hyperpolarizing effects of impulse trains were abolished by the incubation of the nerve fibre in a sea-water solution containing trypsin.6. These findings are discussed in relation to the possible mechanisms that might be responsible for the long-lasting hyperpolarizations of the Schwann cells.     

Associated particle aggregates in juxtaparanodal axolemma and adaxonal Schwann cell membrane of rat peripheral nerve

Summary Freeze-fracture observations have been made on unfixed cryoprotected, and glutaraldehyde-perfused and cryoprotected rat sciatic nerve. In the juxtaparanodal region of the internode, numerous particle clusters were observed on the axolemmal E face and rings of particles of uniform size on the P face of the adaxonal Schwann cell membrane. Both of these particle aggregates were concentrated in the internodal region immediately adjacent to the paranode (juxtaparanodal). The findings provide evidence for a close association between the two particle formations, suggesting a unitary structure forming links between the axolemma and Schwann cell membrane. Figures are given for the density distribution of these particles at the juxtaparanodal region. They were very rarely observed on membrane fracture faces of the general internodal regions. It is possible that these particle formations may represent potassium channels or that they could provide channels for other metabolic communication between the Schwann cell and the axon.     

Immunocytochemical analysis of glial cells in the hypomyelinated optic nerve of the BW mutant rat

Summary The Browman-Wyse (BW) rat is a mutant with structural defects of the visual system, including a failure of the proximal (retinal) end of the optic nerve to myelinate. This latter abnormality is correlated with an absence of CAII+ oligodendrocytes, but we have previously shown that astrocytes are normally distributed, as judged by morphological characteristics of GFAP+ cellsin vivo. We have further examinedin vitro the immunohistochemical characteristics of macroglia isolated from the BW optic nerve, either as cell suspensions or after 4 days in culture.Cell cultures derived from the hypomyelinated proximal segment of BW optic nerves contained very few 0–2A progenitor cells (from which oligodendrocytes and cells with the GFAP+/A2B5+ phenotype develop), whereas over 90% of the glia were Schwann cells. A proportion of these few 0–2A progenitor cells differentiated normally after 4 daysin vitro into both progeny phenotypes in appropriate media. Accordingly, we conclude that the myelination deficiency in the BW optic nerve could be explained as a failure of 0–2A progenitor cells to populate fully the proximal extremity of the nerve during development.Since most glia isolated from adult optic nerves did not adhere to the culture substrate, we analysed the phenotypes of freshly isolated cells in suspension. Comparing optic nerves of normal adult rats with those of BW mutants, a significantly higher fraction of the GFAP+ cells reacted with A2B5 in cell suspensions of the latter. The double-labelled cells which are present in abnormally high numbers may be the differentiated progeny of 0–2A progenitors in the hypomyelinated segment of nerve. One explanation for these findings is that Schwann cells within the BW nerve induce the differentiation of 0–2A progenitor cells to the GFAP+/A2B5+ phenotype. We investigated this possibility using conditioned medium from cultured Schwann cells which increased tenfold the frequency of GFAP+/A2B5+ cells in normal neonatal rat optic nerve cultures. Oligodendrocyte numbers showed a concomitant decline with increasing concentration of Schwann cell conditioned medium.Hypomyelination in the BW rat optic nerve may therefore arise because Schwann cells, present in the proximal segment of the nerve, not only impede the migration of 0–2A progenitor cells but also release a factor which induces those 0–2A progenitor cells which arrive in the proximal segment of the nerve to differentiate into GFAP+ cells at a critical stage in oligodendrocyte development.     

Functional lamination in the ganglion cell layer of the macaque's retina

Close to the fovea of the primate retina the ganglion cell layer is at its maximal thickness and several layers of cells deep. In whole-mount preparations in which the ganglion cells had been retrogradely labelled to reveal the dendritic trees we have studied the distribution of the different ganglion cell types across the depth of the ganglion cell layer. The ganglion cells which project to the parvocellular layers (P ganglion cells) are found more vitread than those which project to the magnocellular layers (M ganglion cells). The cells which project to the midbrain lie in the outer part of the ganglion cell layer among the M cells and adjacent to the inner plexiform layer. Within the P and M classes of ganglion cell the On-centre cells lie more vitread than the Off-centre cells. These results are discussed with relation to the proportions of different cell types sampled with intraocular recordings from ganglion cells and the possible significance for the development of different types of ganglion cell.     

Regeneration of axons in the optic nerve of the adult Browman-Wyse (BW) mutant rat

Summary We have studied the regeneration of axons in the optic nerves of the BW rat in which both oligodendrocytes and CNS myelin are absent from a variable length of the proximal (retinal) end of the nerve. In the optic nerves of some of these animals, Schwann cells are present. Axons failed to regenerate in the exclusively astrocytic environment of the unmyelinated segment of BW optic nerves but readily regrew in the presence of Schwann cells even across the junctional zone and into the myelin debris filled distal segment. In the latter animals, the essential condition for regeneration was that the lesion was sited in a region of the nerve in which Schwann cells were resident. Regenerating fibres appeared to be sequestered within Schwann cell tubes although fibres traversed the neuropil intervening between the ends of discontinuous bundles of Schwann cell tubes, in both the proximal unmyelinated and myelin debris laden distal segments of the BW optic nerve. Regenerating axons never grew beyond the distal point of termination of the tubes. These observations demonstrate that central myelin is not an absolute requirement for regenerative failure, and that important contributing factors might include inhibition of astrocytes and/or absence of trophic factors. Regeneration presumably occurs in the BW optic nerve because trophic molecules are provided by resident Schwann cells, even in the presence of central myelin, oligodendrocytes and astrocytes. All the above experimental BW animals also have Schwann cells in their retinae which myelinate retinal ganglion cell axons in the fibre layer. Control animals comprised normal Long Evans Hooded rats, BW rats in which both retina and optic nerve were normal, and BW rats with Schwann cells in the retina but with normal, i.e. CNS myelinated, optic nerves. Regeneration was not observed in any of the control groups, demonstrating that, although the presence of Schwann cells in the retina may enhance the survival of retinal ganglion cells after crush, concomitant regrowth of axons cut in the optic nerve does not take place.     

Voltage gradients across the receptor layer of the isolated rat retina

1. The electroretinogram (e.r.g.) of the isolated rat retina has been investigated by recording potential differences developed between two micropipettes.

2. In the uniformly illuminated receptor layer, voltage gradients at 90° to the long axes of the receptors are negligible in comparison with the radial voltage gradients.

3. When all transsynaptic neural activity has been abolished, the photoresponse recorded across the receptor layer is very different from the photoresponse recorded across the inner retinal layer.

4. The photoresponse developed across the inner retinal layers, slow P III, develops slowly and the peak voltage is approximately proportional to log. flash energy.

5. The photovoltage across the receptor layer rises rapidly to its peak, before a significant fraction of slow P III has developed.

6. The faster photovoltage (receptor potential) increases with flash intensity according to the hyperbolic function characteristic of photo-receptors.

7. The faster photovoltage can be split into two components. Between the tips of the outer limbs and the bases of the inner limbs, it has a simple wave form. In the region between the bases of the inner limbs and the receptor synapses, there is an additional peak (nose) to the photovoltage.

8. In the scleral portion of the receptor layer, the photovoltage approximately equals the dark voltage. In the remaining, vitreal portion of the receptor layer the photovoltage exceeds the dark voltage.

9. Photocurrent divergence has been measured and the results indicate that the source of photocurrent extends further vitreally than the base of the outer limb.

10. The results suggest that the photoresponse generated in the outer limb is modified by an active process which occurs in portions of the rods which are nearer the synapse.