Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells

To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of l-2,3-[³H]proline into newly-synthetized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2–36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Near-normal levels were recovered by 36 days. In contralateral control neurons, the incorporation of l-2,3-[³H]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 55% at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.     

Removal of optic tectum prolongs the cell body reaction to axotomy in goldfish retinal ganglion cells

Injury to the optic axons of goldfish elicits dramatic changes in the cell bodies of the neurons from which these axons arise, the retinal ganglion cells. The changes include a large increase in cell size and in synthesis and axonal transport of protein. The cells begin to return to normal about 3 weeks after the injury, when the axons invade the contralateral (homotopic) lobe of the optic tectum, and recovery is essentially complete by 8-10 weeks after the lesion. However, if the homotopic lobe of the tectum was removed at the time of nerve crush, we found that the cell body reaction was greatly prolonged. The cells remained enlarged, and [3H]proline incorporation and fast axonal transport of protein remained elevated, until at least 10-12 weeks after nerve crush, although by this time most of the regenerating axons had probably regained their normal length and many had entered the remaining ipsilateral (heterotopic) lobe of the tectum. The cells showed partial recovery by the latest time tested, 26 weeks after nerve crush, when the projections from the two eyes had segregated into separate bands in the heterotopic tectal lobe.     

Protein synthesis and axonal transport in goldfish retinal ganglion cells during regeneration accelerated by a conditioning lesion

Axonal outgrowth in goldfish retinal ganglion cells following a testing lesion of the optic axons is accelerated by a prior conditioning lesion. Changes in protein synthesis and axonal transport were examined during the accelerated regeneration. The conditioning lesion was an optic tract cut made 2 weeks prior to the testing lesion, which consisted of a tract cut at the chiasma, so that nerves subjected to either a conditioning lesion (‘conditioned nerves’) or a sham operation (‘sham-conditioned nerves’) could be examined in the same animal. In the retinal ganglion cells of conditioned nerves, the incorporation of [³H]proline into protein began to increase between 1 and 8 days after the testing lesion. The amount of fast-transported labeled protein was elevated to about 8 × normal by 1 day after the testing lesion but had decreased to about 3–5 × normal at 8 and 22 days. The 8 and 22 day values were not significantly different from those in sham-conditioned nerves or nerves that had received a testing lesion alone. For slow protein transport, the instantaneous amount transported was 15–16 × normal in the conditioned nerves at 1 and 8 days after the testing lesion, and the velocity of slow transport, which was already elevated above normal by 1 day after the testing lesion, was elevated still further by 8 days — to a value in excess of 1.5 mm/day (compared to 0.2–0.4 mm/day in normal animals). We believe that the enhanced outgrowth resulting from the conditioning lesion is due to a transient increase in the amount of fast transport (possibly responsible for a decreased delay in the initiation of sprouting), and a sustained increase in the amount and velocity of slow transport (which may account for an increased rate of elongation).     

Ganglioside changes in the chicken optic lobes as biochemical indicators of brain development and maturation

The development profiles of 16 different gangliosides of the optic lobes of the chicken were followed from the sixth day of incubation to the tenth posthatching week and correlated to known morphological development. Several, previously undetected novel fractions occurred between the sixth and tenth embryonic days. According to their migration rates on TLC-plates 4 of them may be GT3, GT2, GT1c, GQ1c. Three even more slowly migrating fractions represent penta-, hexa-, and septa-sialogangliosides. At the sixth day of incubation, characterized by maximal proliferation of neuro-epithelial cells, the optic lobes contained predominantly GD3.Up to the eleventh day of incubation, parallel to decreased mitotic activity, maximal cell migration and neuron differentiation, GD3, GD2, and GT3 decreased in favor of newly detected polysialogangliosides. Thereafter, up to hatching, parallel to increased growth and arborization of dendrites and axons as well as synaptogenesis, the newly detected polysialogangliosides decreased in favor of GD1b, GT1b, GQ1b, and GD1a.At hatching the myelin-specific GM4 appeared, reaching about 8% of total ganglioside sialic acid after 10 weeks. Likewise a fraction, migrating somewhat faster than GM1,increased. This band, named GM1', is suggested to be also myelin-associated. The other monosialogangliosides were always minor fractions, none exceeding 4% of total ganglioside sialic acid.     

Effects of immunosuppression on regrowth of adult rat retinal ganglion cell axons into peripheral nerve allografts

Analysis of the effectiveness of allografts and immunosuppression in the repair of nerve defects in the adult peripheral nervous system (PNS) has a long experimental and clinical history. There is little information, however, on the use of allografts in peripheral nerve (PN) transplantation into the injured central nervous system (CNS). We assessed the ability of PN allografts (from Dark-Agouti rats) to support regeneration of adult rat retinal ganglion cell (RGC) axons in immunosuppressed host Lewis rats. PN allografts were sutured onto intraorbitally transected optic nerves. Three weeks after grafting, regenerating RGC axon numbers were determined using retrograde fluorescent labelling, and total axons within PN grafts were assessed using pan-neurofilament immunohistochemistry. In the absence of immunosuppression, PN allografts contained few axons and there were very few labelled RGC. These degenerate grafts contained many T cells and macrophages. Systemic (intraperitoneal) application of the immunosuppressants cyclosporin-A or FK506 reduced cellular infiltration into allografts and resulted in extensive axonal regrowth from surviving RGCs. The average number of RGCs regenerating axons into immunosuppressed allografts was not significantly different from that seen in PN autografts in rats sham-injected with saline. Many pan-neurofilament-positive axons, a proportion of which were myelinated, were seen in immunosuppressed allografts, particularly in proximal regions of the grafts toward the optic nerve-PN interface. This study demonstrates that PN allografts can support axonal regrowth in immunosuppressed adult hosts, and points to possible clinical use in CNS repair.     

Pathfinding by retinal ganglion cell axons: Transplantation studies in genetically and surgically blind mice

Optic axons show a highly stereotypical intracranial course to attain the visual centers of the brainstem. Here we examine the course followed by axons arising from embryonic retinae implanted in neonatal ocular retardation mutant mice in which there had been no prior innervation of the visual centers. Retinae placed on the ventrolateral brainstem adjacent to the normal site of the optic tract send axons dorsolaterally toward the ipsilateral superior colliculus, which they innervate along with a number of other subcortical visual centers. Somewhat unexpectedly, axons also course ventrally to cross at the level of the suprachiasmatic nucleus or, less frequently, caudal to the mammillary body to follow the route of the optic tract and innervate contralateral visual centers. Retinae implanted along the course of the internal capsule emit axons that follow projection fibers through the striatum to innervate the lateral geniculate nucleus and other optic nuclei. These grafts also appear to project to the lateral part of the ventrobasal nucleus of the thalamus. The results show that prior existence of an optic projection is not necessary for axons derived from ectopic retinae to attain visual nuclei, not only on the side of implantation but also on the contralateral side of the brain. The cues that these growing axons follow appear to be stable temporally. The fact that axons can also follow highly anomalous routes, such as through the internal capsule, to attain target nuclei in the brainstem suggests that the normal optic pathway is not an obligatory route for optic outgrowth. © 1995 Wiley-Liss, Inc.     

Intraocular injection of tetrodotoxin in goldfish decreases fast axonal transport of [H]glucosamine-labeled materials in optic axons

When physiological activity in goldfish visual system was abolished by repeated intraocular injection of tetrodotoxin (TTX), the fast axonal transport of radioactive amino acid-labeled protein in the optic axons was unaltered. However, the TTX treatment reduced the amount of [³H]glucosamine-labeled glycolipids that were axonally transported to the optic tectum, and may have decreased their rate of turnover in the tectum. A similar though smaller effect was observed for glucosamine-containing glycoproteins. These alterations in axonal transport may be the basis for at least some of the deleterious effects of TTX on axonal regeneration in this system.     

The composition and organization of axonally transported proteins in the retinal ganglion cells of the guinea pig

We labeled the proteins of guinea pig retinal ganglion cells with [³⁵S]methionine and analyzed the axonally transported polypeptides by means of sodium dodecyl sulfate gel electrophoresis. Five groups of transported polypeptides could be distinguished by their characteristic times of initial appearance in segments of the axons of the retinal ganglion cells. The times of initial appearance of the groups corresponded to maximum transport velocities ranging from greater than 200 mm/day to 0.5 mm/day. We directly compared these transported polypeptides to polypeptides undergoing axonal transport in the retinal ganglion cells of the rabbit. Electrophoretically similar polypeptides were transported at the same relative velocities in the two animals. Our results lead to the following conclusions.

(1) The basic composition and organization of axonally transported proteins is probably a general constant feature of mammalian retinal ganglion cells, implying that the correct organization is important for the proper functioning of these neurons. Therefore, the results obtained by the analysis of individual model systems should have general significance.

(2) Four discontinuities in the transport process (in addition to the 5 discontinuities represented by the major transport groups) were revealed by a consideration of subtle differences between the rabbut and guinea pig, as well as differences in the rate of disappearance of label from individual polypeptides within each transport group.

(3) The guinea pig should provide a useful model system for studying axonal transport, especially for immunological studies, since antibodies against axonally transported proteins of the guinea pig can be conveniently prepared in the rabbit.

(4) While the structure (as reflected by electrophoretic mobility) of most major axonally transported polypeptides appears to be conserved over the evolutionary period (about 30 million years) separating two orders of mammals, the electrophoretic mobility of one neurofilament-associated polypeptide, H, was abnormally variant between the two species.

Localization of axonally transported label in chick retinal ganglion cell axons after intravitreal injections of wheat germ agglutinin conjugated to horseradish peroxidase

We have studied the subcellular localization of peroxidase-labeled organelles after anterograde axonal transport by chick retinal ganglion cells that had been exposed 23-25 h earlier to wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After intravitreal injection of WGA-HRP, we found in the optic tectum that 82% of labeled organelles were located within axons and axon terminals. The organelles included: tubules and cisternae of the smooth endoplasmic reticulum, hypolemmal cisternae, vesicles, dense bodies and multivesticular bodies. We also measured the distances between the centers of the labeled organelles and the plasma membrane of these profiles. The density of organelles (number of organelles/micron 2) was plotted as a function of distance from the plasma membrane. Irrespective of the dose of lectin-peroxidase injected, labeled organelles were most densely concentrated in a 30 nm wide annular zone centered 75 nm in from the plasma membrane. In axon terminals the labeled organelles were most concentrated 75-90 nm in from the plasma membrane. Assuming that the peroxidase label indicates the presence of WGA-HRP, we conclude that after anterograde axonal transport the lectin accumulates in lysosomal organelles and elements of the smooth endoplasmic reticulum. Therefore, in contrast to the more restricted localization of [125I]WGA as inferred from electron microscopic autoradiography after uptake and transport by the same cell type, WGA-HRP-labeled organelles are found more diffusely within the axoplasm, particularly in axon terminals. Furthermore, peroxidase-labeled organelles in dendritic, glial or neuronal cell bodies in the tectum were seen less frequently than expected based on evidence of frequent transfer to second cells after intravitreal injections of [125I]WGA. Thus, we infer that at these concentrations WGA labeled with HRP may not be transferred intercellularly as efficiently as even lower concentrations of iodinated WGA are apparently transferred.     

Dorsotemporal retinal ganglion cell axons of goldfish are located in the dorsal rather than ventral optic tract

Cobaltous-lysine was injected into the eyes of goldfish after a slit was made in the temporal retina. Cobalt-filled optic fibers were found in the dorsal optic tract and tracing them to their destinations revealed that they terminated rostrally in the peripheral edges of both the dorsal and ventral aspects of the optic tectum. Hence, axons from ganglion cells in the dorsotemporal retina are in the dorsal optic brachium rather than in the ventral optic brachium as was previously assumed.     

Extra-axonal diffusion in the rabbit optic system: A caution in axonal transport studies

The hazards of using optic nerve (as opposed to optic tract and more distal components of the optic system) to study axonal transport were highlighted by observing the fate of [14C]serine and [3H]glycerol injected into the rabbit eye. Despite prior blockage of axonal transport with colchicine, appreciable radioactivity rapidly appeared in the optic nerve adjacent to the injected eye. Radioactivity decreased exponentially along the entire optic chiasm. Counts were distributed among the lipid, protein, and acid-soluble fractions. Separation of optic nerve lipids revealed appreciable labeling of most lipid classes including those characteristic of myelin; a markedly different labeling pattern was observed for axonally transported lipids. The data are consistent with a mechanism involving extra-axonal diffusion of precursor into the surrounding glia followed by incorporation into lipids and proteins of those cells and ultimately myelin. The phenomenon is discussed in relation to possible errors that were made in interpreting earlier experiments.     

Selective innervation of the goldfish suprachiasmatic nucleus by ventral retinal ganglion cell axons

The contribution of central, peripheral, dorsal, ventral, nasal and temporal retinal ganglion cells to the innervation of the suprachiasmatic nucleus of goldfish was examined with cobaltous-lysine. The nucleus is innervated by axons from central, peripheral, temporal and nasal retina. However, this innervation originates only from ventral retinal ganglion cell axons. The retinal origin of the innervation may be related to being in an aquatic environment.     

Diameters and terminal patterns of retinofugal axons in their target areas: an HRP study in two teleosts (Sebastiscus and Navodon)

Studies in various vertebrate classes, particularly amphibians and mammals, have revealed that retinal ganglion cells with different functional properties project by means of axons of correspondingly different diameters onto specific target regions. Whether a similar pattern exists in teleosts is partly investigated in the present study. HRP was injected into the optic nerve of Sebastiscus and Navodon. The calibers of intraretinal HRP-labeled axons were classed as fine (ca. 0.8 micron), medium (ca. 1.3 micron), and coarse (ca. 2.5 microns). The calibers of HRP-labeled retinofugal axons were then determined in their target areas, and these can be summarized as follows: Optic hypothalamus: fine, medium. Lateral geniculate nucleus: fine. Dorsolateral thalamic nucleus: fine, medium. Area pretectalis: fine. Nucleus of the posterior commissure: fine, medium. Area ventralis lateralis, contralateral: fine, medium, coarse; ipsilateral: coarse. Optic tectum, stratum opticum: fine, medium; stratum fibrosum et griseum superficiale: fine, medium, coarse, segregated in sublayers; stratum album centrale: fine, medium, coarse. Therefore, fine fibers were found to reach all target areas except the ipsilateral area ventralis lateralis, and these were the only fibers found in the lateral geniculate nucleus, area pretectalis, and stratum griseum centrale of the optic tectum. Coarse fibers, on the other hand, were found only in the area ventralis lateralis and the optic tectum (stratum fibrosum et griseum superficiale and stratum album centrale). Terminal patterns of these fibers were also studied. Most fine fibers take tortuous courses giving off a few branches and terminate with many varicosities, and medium and coarse fibers give off several finer branches and terminate with bulbous swellings. The physiological significance of these findings is discussed. In addition, retrogradely labeled (retinopetal) cells were found in the olfactory bulb and the area ventralis pars ventralis of the telencephalon, as well as in the preoptic area and the dorsolateral thalamic nucleus.     

Coexpression of lactosyl and gangliotetraosyl gangliosides in rat cerebellar radial glial cells in culture

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. To distinguish the major neural cell types present in the cultures the expression of the following cell type-specific markers was investigated: neuron-specific enolase and microtubule-associated protein-2 (MAP-2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells. More than 80% of cells dissociated from cerebellar tissue of 15-day-old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl-GTC, but most are negative for the expression of neuron-specific enolase, MAP-2, and GFAP. After culturing for 4 days (E15 + 4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl-GTC and "inactivate" the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl-GTC, but unlike neuron-like cells, they do not "inactivate" the expression of GD3.     

Spatial correspondence between R-cadherin expression domains and retinal ganglion cell axons in developing zebrafish.

Mechanisms underlying axonal pathfinding have been investigated for decades, and numerous molecules have been shown to play roles in this process, including members of the cadherin family of cell adhesion molecules. We showed in the companion paper that a member of the cadherin family (zebrafish R-cadherin) is expressed in retinal ganglion cells, and in presumptive visual structures in zebrafish brain, during periods when the axons were actively extending toward their targets. The present study extends the earlier work by using 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) anterograde tracing techniques to label retinal ganglion cell axons combined with R-cadherin in situ hybridization to explicitly examine the association ofretinal axons and brain regions expressing R-cadherin message. We found that in zebrafish embryos at 46-54 hours postfertilization, DiI-labeled retinal axons were closely associated with cells expressing R-cadherin message in the hypothalamus, the pretectum, and the anterolateral optic tectum. These results demonstrate that R-cadherin is appropriately distributed to play a role in regulating development of the zebrafish visual system, and in particular, pathfinding and synaptogenesis of retinal ganglion cell axons.     

The effect of light exposure following an intraocular injection of [H]N-acetylmannosamine on the labeling on gangliosides and glycoproteins of retina ganglion cells and optic tectum of singly caged chickens

Ten-day-old chickens that after a 2-day-period of adaptation to dark received an intraocular injection of [³H]N-acetylmannosamine ([³H]ManNAc) and were exposed, individually housed, to light, have more labeling in the gangliosides and glycoproteins of the ganglion cell layer of retina and in the contralateral optic tectum compared to their counterparts that remained in darkness. No differences were found in the labeling of the acid soluble fraction of the ganglion cell layer between the animals in dark and light at 0.5 and 5 h after the injection of [³H]ManNAc.No differences could be observed in the quality of storage of the gangliosides labeled in light with respect to those labeled in dark, but those labeled in light had a higher percent of labeling released by neuraminidase at 5 h after the intraocular injection of the labeled precursor.In animals exposed to intermittent light, the increased labeling with respect to dark was smaller than that found in animals exposed continously to light.     

Transport of cytoskeletal proteins in axons of hippocampal pyramidal cells

Axonal transport of cytoskeletal proteins has not yet been extensively studied in the brain proper, in contrast to the peripheral nerves and optic nerves. The authors have developed a means for the study of transport of cytoskeletal proteins in axons of hippocampal pyramidal cells. Proteins of intrinsic neurons of the dorsal hippocampus were labeled by microinjection of ³⁵S methionine, and the subsequent transport of labeled proteins was characterized in the axons projecting into the fimbria-fornix. A peak of labeled proteins was present in the fimbria-fornix at 4–12 days after labeling, corresponding to transport rates 0.2–0.7 mm/day. The most abudant proteins at each time studied exhibited one-dimensional electrophoretic mobilites of actin and tubulin; neurofilaments were less intensely labeled. The observed specializations of cytoskeletal transport, especially the paucity of tubulin transport at rates of 2–4 mm/day, may predispose hippocampal pyramidal cells to accumulate tubulin and microtubule-associated proteins in their cell bodies in various disease states.     

Anterograde and retrograde axonal transport of native and derivatized wheat germ agglutinin in the visual system of the chicken

The anterograde and retrograde rates of axonal transport of the lectin wheat germ agglutinin (WGA) were investigated using native and derivatized lectins in embryonic (stage 39) and posthatch chickens. Anterograde transport rates in the retinotectal projection of posthatch animals ranged from 168 mm/day for native WGA to 345 mm/day for horseradish peroxidase conjugated WGA. Anterograde transport rates in embryos were at least 258 mm/day based on experiments employing tritium and horseradish peroxidase conjugates. Retrograde rates measured by appearance of label in the isthmo-optic nucleus in both embryonic and posthatch chickens were in the range of 150-180 mm/day. A fluorescein isothiocyanate conjugate of WGA was transported retrogradely but not anterogradely. When the extraocular eye muscles were labeled accidentally during injection, cells in the oculomotor or trochlear nuclei were more intensely labeled than neurons in the isthmo-optic nucleus. It was concluded that at least some conjugates of WGA, and possible the native lectin as well, travel in the fastest component of axonal transport. In view of the known intercellular movement of WGA from labeled presynaptic processes, it is recommended that survival times be kept short in experiments using WGA as a neuronal tracing agent (less than 24 h) to minimize the possibility of uptake and redistribution of the lectin by nearby cells.     

Ultrastructural evidence of the formation of synapses by retinal ganglion cell axons in two nonstandard targets

After unilateral ablation of the optic tectum in the frog (Rana pipiens), retinal ganglion cell axons enter the lateral thalamic neuropil in large numbers. This area is normally a target of the tectal efferent projection but is not innervated directly from the retina in normal frogs nor in frogs undergoing optic nerve regeneration in the presence of an intact tectum. The ability of retinal axons to form synaptic contacts in this nonstandard target, previously suspected only from light microscope studies, has been ultrastructurally verified in the present investigation. Retinal axon terminals were selectively labeled for light and electon microscope study by introducing horseradish peroxidase (HRP) into the optic nerve 73-413 days after unilateral ablation of the contralateral optic tectum. In some of the frogs, the optic nerve had also been crushed to test the ability of retinal axons regenerating over a long distance to form this connection. The HRP-labeled retinal axon terminals had the same untrastructural morphology whether located in the lateral thalamic neuropil or in the correct regions of projection, e.g., the lateral geniculate complex. They contained clear, spherical synaptic vesicles and made Gray type I synapses on the unlabeled postsynaptic dendrites. The magnitude of the projection was disproportionately greater in animals having complete or nearly complete tectal ablation than in a specimen in which the lesion was significantly incomplete. An aberrant projection was also observed in the nucleus isthmi in some of the specimens. These findings have significance for chemoaffinity theories of the specification of synaptic connections in that the ability of retinal axons to synapse in nonstandard targets in this experimental context may be considered evidence for the expression of appropriate cell-surface recognition-molecules by the abnormally targeted postsynaptic neurons. The likelihood that the expression of these postsynaptic labels is normally repressed transynaptically by molecular signals from the intact tectal input is discussed.