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.

Voltage-gated sodium and potassium channels in radial glial cells of trout optic tectum studied by patch clamp analysis and single cell RT-PCR.

Radial glial cells in the visual center of trout were analyzed immunocytochemically and with the whole cell mode of the patch-clamp technique in combination with RT-PCR. By immunostaining with anti-GFAP antibodies radially oriented cell processes spanning the entire width of the tectum were brightly labeled, while with anti-S-100 antiserum the cell bodies residing in a discrete layer close to the ventricular border became most clearly visible. Virtually all radial glial cells examined in brain slices exhibited voltage-gated sodium inward currents that were activated above -40 mV, blocked by micromolar concentrations of TTX and totally eliminated if sodium was substituted for Tris in the bath solution. In contrast with adjacent nerve cells of the same slices radial glial cells did not exhibit spontaneous electrical activity and could not be stimulated to generate action potentials by depolarizing current injections. Two types of voltage-gated potassium outward currents were elicited by depolarizing voltage steps: a sustained current with delayed rectifier properties and a superimposed transient "A"-type current, both being activated at a threshold potential of -40 mV. In cultured radial glial cells subtle differences were noticed regarding current density, inactivation kinetics, and TEA-sensitivity of the potassium currents. Inwardly rectifying potassium currents activating at hyperpolarized voltages were not observed. By single cell RT-PCR the transcripts of two shaker-related potassium channel genes (termed tsha1-a fish homologue to Kv1.2- and tsha3) were amplified, while transcripts for tsha 2 and tsha 4 were not detected.

Temporary colonization of the site of lesion by macrophages is a prelude to the arrival of regenerated axons in injured goldfish optic nerve.

In crushed goldfish optic nerve, regenerating axons cross the site of lesion within 10 days following injury. Some 30 days later, Schwann cells accumulate at the lesion, where they myelinate the new axons. In this study, we have used immunohistochemistry and electron microscopy to examine the cellular environment of the crush site prior to the establishment of Schwann cells in order to learn more about the early events that contribute to axonal regeneration. During the first week following injury, macrophages enter the site of lesion and efficiently phagocytose the debris. The infiltration of macrophages precedes the arrival of regenerating axons that abut and surround these phagocytes. Based on EM morphology and phagocytic capacity, macrophages of the type observed at the site of lesion are not present in the degenerating distal nerve segment, where debris clearance is shared between conventional microglia and astrocytes over a period of several weeks. During this period, axon bundles emerging distally from the injury zone become enwrapped by astrocyte processes, thereby re-establishing the characteristic fascicular cytoarchitecture of the optic nerve. The process of fasciculation also leads to the displacement of myelin debris to the margins of the fiber bundles, where it is trapped by the astrocytes. Our results suggest that the early robust appearance of macrophages at the lesion, and their effectiveness as phagocytes compared with the microglia distally, may contribute to the vigorous axonal regeneration across the crush, beyond which axons--excepting the pioneers--extend through newly formed debris-free channels delineated by astrocyte processes.

Glial repair at the lesion site in regenerating goldfish spinal cord: an immunohistochemical study using species-specific antibodies.

We have used fish-specific antibodies to show that repair in regenerating goldfish spinal cord is accompanied by the recovery of the astrocytic environment and restoration of the central canal. Astrocyte processes trailed the regenerated axons bridging the new cord, suggesting that they are not needed for axonal regrowth.

Myelin repair by Schwann cells in the regenerating goldfish visual pathway: regional patterns revealed by X-irradiation.

In the regenerating goldfish optic nerves, Schwann cells of unknown origin reliably infiltrate the lesion site forming a band of peripheral-type myelinating tissue by 1-2 months, sharply demarcated from the adjacent new CNS myelin. To investigate this effect, we have interfered with cell proliferation by locally X-irradiating the fish visual pathway 24h after the lesion. As assayed by immunohistochemistry and EM, irradiation retards until 6 months formation of new myelin by Schwann cells at the lesion site, and virtually abolishes oligodendrocyte myelination distally, but has little or no effect on nerve fibre regrowth. Optic nerve astrocyte processes normally fail to re-infiltrate the lesion, but re-occupy it after irradiation, suggesting that they are normally excluded by early cell proliferation at this site. Moreover, scattered myelinating Schwann cells also appear in the oligodendrocyte-depleted distal optic nerve after irradiation, although only as far as the optic tract. Optic nerve reticular astrocytes differ in various ways from radial glia elsewhere in the fish CNS, and our observations suggest that they may be more permissive to Schwann cell invasion of CNS tissue.

Myelination of regenerated axons in goldfish optic nerve by Schwann cells.

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.

A polyclonal antibody to goldfish neuronal 145 kDa intermediate filament protein.

A polyclonal antibody raised to a protein from goldfish optic tectum recognises, immunohistochemically, axons throughout normal goldfish visual pathway. In goldfish with injured optic nerve, this antibody recognises degenerating neuronal debris as well as regenerating fibres. On immunoblot, the antibody recognises, primarily, a neuronal intermediate filament protein in the region of 145 kDa. Such an antibody should prove useful in studies pertaining to goldfish visual pathway.

Preferential histochemical staining of protoplasmic and fibrous astrocytes in rat CNS with GFAP antibodies using different fixatives.

Serial sections of rat brain and spinal cord were fixed in either acid-alcohol or 4% paraformaldehyde, and stained for visualization of astrocytes using GFAP antibodies. With paraformaldehyde, GFAP-positive astrocytes were visualised almost exclusively in the grey matter of all above tissues. In sharp contrast, acid-alcohol treatment gave intensely stained GFAP-containing astrocytes in the white matter. Since fibrous astrocytes are mainly located in the white matter and protoplasmic astrocytes are located in the grey matter, it is concluded that acid-alcohol is a good fixative for fibrous astrocytes while paraformaldehyde is a better fixative for protoplasmic astrocytes.

Expression of glial fibrillary acidic protein (GFAP) in goldfish optic nerve following injury.

By using an antibody to goldfish glial fibrillary acidic protein (GFAP), the reaction of goldfish optic nerve to injury has been studied by immunoblotting and immunohistochemical methods. Goldfish optic nerve, which normally lacks GFAP immunoreactivity (Nona et al.: Glia, 2:189-200, 1989), expresses GFAP following injury. This immunoreactivity, which is observed as early as 10 days after crush and which is still evident at 30 days after crush, all but disappears by 150 days after crush. Since it is well established that functional restoration of synaptic connections and the recovery of vision takes place in goldfish following optic nerve injury, our results indicate that reactive astrocytes do not represent an impediment to regeneration in goldfish visual system.

Anti-goldfish glial fibrillary acidic protein (GFAP) recognises astrocytes from rat CNS.

A polyclonal antibody to goldfish GFAP recognises, immunohistochemically, astrocyte populations in rat brain, spinal cord and optic nerve. The pattern of staining compares favourably with that obtained using a polyclonal anti-human GFAP or a monoclonal anti-porcine GFAP. These results are consistent with the notion that GFAP is well conserved in vertebrate phylogeny.

Glial fibrillary acidic protein (GFAP) from goldfish: its localisation in visual pathway.

An intermediate filament fraction, isolated from goldfish brain, contains a prominent protein having a molecular weight of 51 kDa. In normal goldfish visual pathway, this protein is present in tectum and tract, but not in optic nerve. A polyclonal antibody raised to this protein clearly labels ependymal glial profiles in tectum and parallel processes in the tract, whereas optic nerve is unlabelled; Müller fibres in the retina are also labelled. A similar, but less prominent, pattern of staining is observed with antibodies, raised elsewhere, against glial fibrillary acidic protein from human and porcine. These results suggest that the 51 kDa protein is a GFAP, demonstrate the heterogeneity of astrocytes in goldfish visual pathway, and are consistent with the idea that GFAP is well conserved in vertebrate phylogeny.

Postnatal developmental profiles of filamentous actin and of 200 kDa neurofilament polypeptide in the visual cortex of light- and dark-reared rats and their relationship to critical period plasticity.

The concept of critical period plasticity in rat visual cortex has been studied in terms of changes in the level of filamentous actin and of the 200 kDa neurofilament polypeptide. Our results suggest that the postnatal developmental profile of filamentous actin is affected by visual experience, as a consequence of eye-opening. No such correlation, however, is detected for the 200 kDa neurofilament polypeptide. The significance of these findings in relationship to neuronal plasticity is discussed in terms of changes in the state and equilibrium conditions of the cytoskeletal proteins.