Axons In Goldfish Research Articles

Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, April 29???May 4, 2001

Association for Research in Vision and Ophthalmology (ARVO) Annual Meeting, April 29???May 4, 2001

Fate of oligodendrocytes during retinal axon degeneration and regeneration in the goldfish visual pathway

Retinal axons in goldfish regenerate after optic nerve lesion, restore synaptic connections, and become myelinated by oligodendrocytes. The fate of oligodendrocytes during these events is not known and may require generation of new oligodendrocytes or dedifferentiation and redifferentiation of the existing ones. To determine the reaction of oligodendrocytes to optic nerve lesion, we used the terminal transferase technique to detect apoptosis, bromodeoxyuridine incorporation to reveal mitosis, antibodies to identify myelin and oligodendrocytes, and Lucifer yellow injections to reveal cell morphology. Along with the reappearance of the myelin molecules 36K protein, galactocerebroside, and myelin basic protein, myelinating oligodendrocytes (identified by Lucifer yellow injections) reappear 21 days postlesion. Prior to this time, the dye-filled cells had few processes oriented along the regenerating axons. They resembled oligodendrocytes seen both in vitro and in vivo which express the L1-related E587 antigen and synthesize the 36K myelin protein in coculture with axons. No signs of oligodendrocyte apoptosis were detected after lesion and only few of the oligodendrocytes present had recently arisen. 36K/E587 double-labeled oligodendrocytes which were most likely dedifferentiating oligodendrocytes were identified in 8-day postlesion nerves among E587-positive elongate cells whose numbers increased until 14 days postlesion. These findings suggest that oligodendrocytes dedifferentiate-like Schwann cells-from cells which express myelin molecules to elongate cells which express the L1/E587 antigen. They redifferentiate to myelinate axons from roughly 3 weeks onward. These findings suggest an adaptive plasticity of goldfish oligodendrocytes beneficial to the repair of the visual pathway.

Regeneration of ascending spinal axons in goldfish

Regeneration of descending spinal cord tracts occur spontaneously in adult goldfish. Very little information is available regarding the fate of ascending fibers. Using Dextran amines as a tracer, we studied the normal and regenerated ascending axonal projection patterns in adult goldfish brain nuclei. Present study includes spinal projections to torus semicircularis, hypothalamus, thalamus and the telencephalon. Regenerated fibers had finer caliber axons and the terminal axonal arbors covered a larger area than the corresponding normal ones.

Optic nerve glia secrete a low-molecular-weight factor that stimulates retinal ganglion cells to regenerate axons in goldfish

The ability of lower vertebrates to regenerate an injured optic nerve has been widely studied as a model for understanding neural development and plasticity. We have recently shown that, in goldfish, the optic nerve contains two molecules that stimulate retinal ganglion cells to regenerate their axons in culture: a low-molecular-weight factor that is active even at low concentrations (axogenesis factor-1) and a somewhat less active polypeptide of molecular weight 10,000-15,000 (axogenesis factor-2). Both are distinct from other molecules described previously in this system. The present study pursues the biological source and functional significance of axogenesis factor-1. Earlier studies have shown that cultured goldfish glia provide a highly favorable environment for fish or rat retinal ganglion cells to extend axons. We report that the glia in these cultures secrete high levels of a factor that is identical to axogenesis factor-1 in its chromatographic properties and biological activity, along with a larger molecule that may coincide with axogenesis factor-2. Axogenesis factor-1 derived from either goldfish glial cultures or optic nerve fragments is a hydrophilic molecule with an estimated molecular weight of 700-800. Prior studies have reported that goldfish retinal fragments, when explanted in organ culture, only extend axons if the ganglion cells had been "primed" to begin regenerating in vivo for one to two weeks. However, axogenesis factor-1 caused the same degree of outgrowth irrespective of whether ganglion cells had been induced to regenerate new axons in vivo. Moreover, ganglion cells primed to begin regenerating in vivo continued to extend axons in culture only when axogenesis factor-1 was present. In summary, this study shows that glial cells of the goldfish optic nerve secrete a low-molecular-weight factor that initiates axonal regeneration from retinal ganglion cells.

Light microscopic study of degenerating cobalt-filled optic axons in goldfish: role of microglia and radial glia in debris removal.

Labeling severed axons with cobaltous-lysine ultimately leads to the degeneration of their distal segments. The present study was designed to determine whether microglia and radial glia have comparable roles in the elimination of degenerating axons. Another purpose was to determine whether the cobalt could escape from degenerating axons and enter intact neuronal cells. Optic axons were filled with cobaltous-lysine for 1 day and the retinal projections were examined from 1 to 106 days later. Optimal filling was obtained 1 day postlabeling. The number of filled axons in the optic tract was significantly reduced at the 2-day time point, indicating that many axons had disintegrated. Many axons contained large swellings that resembled cells. However, transneuronally labeled neuronal cells were never observed. Labeled, rounded microglia appeared among the degenerating axons at the 3-day time point, and the microglia changed shape at 5 days. They became elongated and manifested many processes. In addition, the microglia began to move toward, and entered, the ventricles and vasculature. Virtually all the labeled debris was removed between 17 and 28 days following the application of cobalt. The rapidity with which the axons were removed suggests that the cobalt accelerates the degenerative process either directly, or indirectly by accelerating the arrival of phagocytic cells. Radial glia appeared to play a smaller role in debris elimination. They took up labeled debris to a lesser degree than microglia and were briefly labeled. Interestingly, radial glia did not take up cobalt when it was injected intracranially and diffused through the brain. A previous claim of an axosomatic retinotectal projection to cells deep in the stratum periventriculare of a teleost fish is reinterpreted to represent cobalt within radial glia.

Pathways of regenerated retinotectal axons in goldfish I. Optic nerve, tract and tectal fascicle layer

This study investigates the order of regenerating retinal axons in the goldfish. The spatiotemporal pattern of axon regrowth was assessed by applying horseradish peroxidase (HRP) to regenerating axons in the optic tract at various times after optic nerve section and by analysing the distribution of retrogradely labelled ganglion cells in retina. At all regeneration stages labelled ganglion cells were widely distributed over the retina. There was no hint that axons from central (older) ganglion cells might regrow earlier, and peripheral (younger) ganglion cells later, as occurs in normal development. The absence of an age-related ordering in the regenerated optic nerve was demonstrated by labelling a few axon bundles intraorbitally with HRP (Easter, Rusoff & Kish, 1981) caudal to the previous cut. The retrogradely labelled cells in retina were randomly distributed in regenerates and not clustered in annuli as in normals. Tracing regenerating axons which were stained anterogradely from intraretinal HRP applications or retrogradely from single labelled tectal fascicles illustrated the fact that the regenerating axons coursed in abnormal routes in the optic nerve and tract. On the surface of the tectum regenerated fibres re-established a fascicle fan. The retinal origin of tectal fascicles was assessed by labelling individual peripheral, intermediate and rostral fascicles with HRP. The retrogradely labelled ganglion cells in the retina were often more widely distributed than in normals, but were mostly found in peripheral, intermediate and central retina, respectively. The order of fibre departure from each tectal fascicle was revealed by placing HRP either on the fascicle's proximal or on its distal half. With proximal labelling sites labelled ganglion cells were found in the temporal and nasal retina, and with distal labelling sites labelled ganglion cells were confined to nasal retina only. Further, the axonal trajectories of anterogradely labelled dorsotemporal retinal ganglion cells were compared to those of dorsonasal retinal ganglion cells in tectal whole mounts. Dorsotemporal axons were confined to the rostral tectal half, whereas dorsonasal axons followed fascicular routes into the fascicle's distal end and reached into caudal tectum. This suggests that the fibres exited along their fascicle's course in a temporonasal sequence. Thus in the tectum, fibres in fascicles restore a gross spatial and age-related order and tend to follow their normal temporonasal sequence of exit.

A regional analysis of alpha-spectrin in the isolated Mauthner neuron and in isolated axons of the goldfish and rabbit

Isolated dendrites, somata, and desheathed axons of the goldfish Mauthner neuron (M-cell), in addition to other isolated myelin sheath-free axons of the goldfish spinal cord and of rabbit lumbar ventral roots, were shown by immunochemical and immunofluorescence techniques to contain alpha-spectrin (fodrin). alpha-Spectrin appeared to be organized as a randomly distributed reticular network, localized to the surface of isolated neuronal cellular structures. In addition, alpha-spectrin was also distributed nonrandomly at specialized cellular sites. These sites included synaptic junctions and morphologically differentiated nodes of Ranvier (i.e., rabbit axons, but not goldfish axons). At the latter sites, it is possible to demonstrate that alpha-spectrin is co-localized with F-actin, as indicated by a striking correspondence of fluorescent images due to double labeling, using the indirect immunofluorescence technique with alpha-spectrin antiserum, and direct binding of F-actin by rhodamine-conjugated palloidin. However, the spectrin-actin network at synaptic junctions appears to be distributed over the entire area of junctional contact and is not just restricted to postsynaptic densities. The possibility of a duality of roles of spectrin in membrane-related motile and anchorage functions is discussed.

Recovery from spinal transection in fish: Regrowth of axons past the transection

In fish axons reportedly cross a spinal transection, form synapses, and then cease growing or grow in abnormal directions. This obviously would not restore previous synaptic connections, and since fish recover function, it is often stated that recovery of function in fish seems to be independent of restoration of normal synaptic architecture. By contrast, the present study emphasizes that many axons in goldfish continue growing after they pass a spinal transection. Horseradish peroxidase was injected into caudal spinal cord in normal or spinal cord-transected fish. Neurons were labeled in the same places in operated and normal fish. Since the site of injection was 4 cm (18 segments) caudal to the transection, it seems clear that the axons of cells labeled in transected fish extended past the cut for almost the length of the spinal cord. Thus growth did not cease for these axons. Therefore, even though synapses undoubtedly form in the neuropil distal to the cut, many axons do not stop growing and restoration of previous synaptic connections may well be a requirement for return of function.

The re-establishment of synaptic transmission by regenerating optic axons in goldfish: Time course and effects of blocking activity by intraocular injection of tetrodotoxin

Intraocular injections of tetrodotoxin were used to block activity for 27 days in normal fish and for the first 27 or 31 days of regeneration in fish with one optic nerve crushed. Synaptic activity was then assessed by a current source-density analysis of field potentials evoked by optic nerve shock at different times following the TTX treatment. In normal fish, the lack of activity for 4 weeks had no significant effect on the maintenance of synaptic strength. Likewise, in fish with nerve crush, lack of activity did not prevent the regenerating optic fibers from forming synapses that were nearly as effective as those formed in controls injected with the citrate buffer vehicle. The earliest synapses were formed at the rostromedial corner of the tectum (where the tract enters) at 20 days after nerve crush, when fibers had not yet reached the caudal areas. By 28 days synaptic potentials could be recorded everywhere on the surface of the tectum in both controls and TTX injected fish. However, the latency of the responseswith TTX were longer, suggesting a smaller caliber of fiber, which is consistent with an earlier finding of decreased axonal transport in TTX fish 6. Maturation of the regenerating fibers proceeded slowly in both TTX and control fish. After more than 5 months, the projections were nearly normal but still not completely normal.

A quantitative study of regenerative sprouting by optic axons in goldfish.

AbstractRegenerative sprouting by optic axons of goldfish was examined distal to the site of a crush lesion. Normally, optic nerves of large goldfish contain approximately 2 × 105 myelinated axons. The number of axonal processes in the nerve distal to the lesion increases fourfold during the first 3 months postoperatively and then returns toward preoperative numbers after 3 months. The excess number of axonal processes therefore persists several months beyond the return of visual function. The magnitude and time course of regenerative sprouting by goldfish optic axons is similar to that reported for mammalian and nonmammalian peripheral axons.

Retinal projection in a non-visual area after bilateral tectal ablation in goldfish.

If, in the adult goldfish, one optic tectum is ablated, the regenerating optic axons from the contralateral retina innervate the remaining tectum, where they form a retinotopically ordered map. The pathway for this induced ipsilateral projection coincides with many of the pathways which normally connect the two tecta, but early in regeneration the optic fibres also enter non-visual centres to which there are degenerating tectal efferent pathways to follow. We have therefore now investigated the fate of regenerating optic axons in goldfish from which both optic tecta have been removed; they are found to innervate non-visual centres, where again they generate a retinotopic map.

Horizontal cell axons and axon terminals in goldfish retina.

Retinas of ordinary and black moor varieties of goldfish (Carassius auratus) were prepared by the Golgi method, mounted flat or sectioned vertically, and studied in the light microscope. Three types of horizontal cells whose dendrites contact only cones, and one type whose dendrites contact only rods, were observed. The cone horizontal cells (Cajal's "external horizontal cells") all have slender axons which descend gradually to the inner nuclear layer and terminate there in long, fusiform expansions (Cajal's "internal horizontal cells"). The thin and thick portions of the axons, as well as the perikarya of the horizontal cells, bear small numbers of straight, horizontally-directed, knobby filamentous appendages which may be sites of synaptic contact. The cone horizontal cell axons in goldfish, unlike those in higher vertebrates, do not terminate in contact with synaptic endings of photoreceptor cells, but in proximity to cells and processes deep in the inner nuclear layer. Axons have not yet been demonstrated on rod horizontal cells in goldfish.